Cyclic di-nucleotide compounds as sting agonists

ABSTRACT

A class of polycyclic compounds of general formula (I), of general formula (I′), or of general formula (I″), wherein Base 1 , Base 2 , Y, Y a , X a , X a1 , X b , X b1 , X c , X c1 , X d , X d1 , R 1 , R 1a , R 2 , R 2a , R 3 , R 4 , R 4a , R 5 , R 6 , R 6a , R 7 , R 7a , R 8 , and R 8a  are defined herein, that may be useful as inductors of type I interferon production, specifically as STING active agents, are provided. Also provided are processes for the synthesis and use of compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/204,677, filed Aug. 13, 2015, U.S. Provisional Patent Application No. 62/268,723, filed Dec. 17, 2015, and U.S. Provisional Patent Application No. 62/356,125, filed Jun. 29, 2016.

FIELD OF THE INVENTION

The present disclosure relates to cyclic di-nucleotide compounds and derivatives thereof that may be useful as STING (Stimulator of Interferon Genes) agonists that activate the STING pathway. The present disclosure also relates to processes for the synthesis and to uses of such cyclic di-nucleotide compounds.

BACKGROUND OF THE INVENTION

The immune system has evolved to recognize and neutralize different types of threats in order to maintain the homeostasis of the host, and it is generally broken down into two arms: adaptive and innate. The adaptive immune system is specialized to recognize antigens not naturally expressed in the host as foreign and to mount an anti-antigen response through the coordinated actions of many leukocyte subsets. The hallmark of adaptive immune responses is their ability to provide “memory” or long-lasting immunity against the encountered antigen. While this specific and long-lasting effect is critical to host health and survival, the adaptive immune response requires time to generate a full-blown response.

The innate immune system compensates for this time delay and is specialized to act quickly against different insults or danger signals. It provides the first line of defense against bacteria, viruses, parasites and other infectious threats, but it also responds strongly to certain danger signals associated with cellular or tissue damage. The innate immune system has no antigen specificity but does respond to a variety of effector mechanisms. Opsonization, phagocytosis, activation of the complement system, and production of soluble bioactive molecules such as cytokines or chemokines are all mechanisms by which the innate immune system mediates its response. By responding to these damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) described above, the innate immune system is able to provide broad protection against a wide range of threats to the host.

Free cytosolic DNA and RNA are among these PAMPs and DAMPs. It has recently been demonstrated that the main sensor for cytosolic DNA is cGAS (cyclic GMP-AMP synthase). Upon recognition of cytosolic DNA, cGAS catalyzes the generation of the cyclic-dinucleotide 2′-3′ cGAMP, an atypical second messenger that strongly binds to the ER-transmembrane adaptor protein STING. A conformational change is undergone by cGAMP-bound STING, which translocates to a perinuclear compartment and induces the activation of critical transcription factors IRF-3 and NF-κB. This leads to a strong induction of type I interferons and production of pro-inflammatory cytokines such as IL-6, TNF-α and IFN-γ.

The importance of type I interferons and pro-inflammatory cytokines on various cells of the immune system has been very well established. In particular, these molecules strongly potentiate T cell activation by enhancing the ability of dendritic cells and macrophages to uptake, process, present and cross-present antigens to T cells. The T cell stimulatory capacity of these antigen-presenting cells is augmented by the up-regulation of critical co-stimulatory molecules, such as CD80 or CD86. Finally, type I interferons can rapidly engage their cognate receptors and trigger the activation of interferon-responsive genes that can significantly contribute to adaptive immune cell activation.

From a therapeutic perspective, type I interferons are shown to have antiviral activities by directly inhibiting human hepatitis B virus and hepatitis C virus replication, and by stimulating immune responses to virally infected cells. Compounds that can induce type I interferon production are used in vaccines, where they act as adjuvants, enhancing specific immune responses to antigens and minimizing side effects by reducing dosage and broadening the immune response.

In addition, interferons, and compounds that can induce interferon production, have potential use in the treatment of human cancers. Such molecules are potentially useful as anti-cancer agents with multiple pathways of activity. Interferons can inhibit human tumor cell proliferation directly and may be synergistic with various approved chemotherapeutic agents. Type I interferons can significantly enhance anti-tumor immune responses by inducing activation of both the adaptive and innate immune cells. Finally, tumor invasiveness may be inhibited by interferons by modulating enzyme expression related to tissue remodeling.

In view of the potential of type I interferons and type I interferon-inducing compounds as anti-viral and anti-cancer agents, there remains a need for new agents that can induce potent type I interferon production. With the growing body of data demonstrating that the cGAS-STING cytosolic DNA sensory pathway has a significant capacity to induce type I interferons, the development of STING activating agents is rapidly taking an important place in today's anti-tumor therapy landscape.

SUMMARY OF THE INVENTION

The present disclosure relates to novel cyclic di-nucleotide compounds of general formula (I), general formula (I′), and/or general formula (I″). In particular, the present disclosure relates to compounds having the general structural formula (I):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as described herein. The present disclosure also relates to compounds having general structural formula (I′):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as described herein. The present disclosure also relates to compounds having general structural formula (I″):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as described herein.

Embodiments of the disclosure include compounds of general formula (I), compounds of general formula (I′), and/or compounds of general formula (I″), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as well as synthesis and isolation of compounds of general formula (I), compounds of general formula (I′), and/or compounds of general formula (I″), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. Uses of compounds of general formula (I), compounds of general formula (I′), and/or compounds of general formula (I″) are also disclosed.

Other embodiments, aspects and features of the present disclosure are either further described in or will be apparent from the ensuing description, examples and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure includes compounds of general formula (I), compounds of general formula (I′), and/or compounds of general formula (I″) above, and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. These compounds and their pharmaceutically acceptable salts, hydrates, solvates, and/or prodrugs are useful as agents to induce interferon production.

A first embodiment of the disclosure relates to cyclic di-nucleotide compounds of general formula (I):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of—O—, —S—, —SO₂—, —CH₂—, and —CF₂—; X^(a) and X^(a1) are each independently selected from the group consisting of O, C, and S; X^(b) and X^(b1) are each independently selected from the group consisting of O, C, and S; X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R⁴ and R⁵ are connected to form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.

In specific aspects of this embodiment, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and

Base¹ and Base² are each selected from the group consisting of R⁵ and R³ are not both selected from the group consisting of H, F and OH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

either only one of R⁵ and R³ is selected from the group consisting of H, F, and OH, or neither R⁵ and R³ is selected from the group consisting of H, F, and OH. In further specific instances of this aspect, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆ alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² are each selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the first embodiment, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In particular instances, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In even more particular instances, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above.

In a second aspect of the first embodiment, Y and Y^(a) are each independently selected from the group consisting of —O— and —S—. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first aspect described above.

In a third aspect of the first embodiment, X^(a) and X^(a1) are each independently selected from the group consisting of O and S. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through second aspects described above.

In a fourth aspect of the first embodiment, X^(b) and X^(b1) are each independently selected from the group consisting of O and S. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through third aspects described above.

In a fifth aspect of the first embodiment, X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹, where each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In particular instances, X^(c) and X^(c) are each independently selected from the group consisting of O⁻, S⁻,

In all instances of this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourth aspects described above.

In a sixth aspect of the first embodiment, X^(d) and X^(d1) are each independently selected from the group consisting of O and S. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fifth aspects described above.

In a seventh aspect of the first embodiment, R¹ and R^(1a) are each H. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through sixth aspects described above.

In an eighth aspect of the first embodiment, R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through seventh aspects described above.

In a ninth aspect of the first embodiment, R³ is selected from the group consisting H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R³ are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through eighth aspects described above.

In a tenth aspect of the first embodiment, R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through ninth aspects described above.

In an eleventh aspect of the first embodiment, R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R⁵ are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through tenth aspects described above.

In a twelfth aspect of the first embodiment, R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through eleventh aspects described above.

In a thirteenth aspect of the first embodiment, R⁷ and R^(7a) are each H. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through twelfth aspects described above.

In a fourteenth aspect of the first embodiment, R⁸ and R^(8a) are each H. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through thirteenth aspects described above.

In a fifteenth aspect of the first embodiment, R^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a sixteenth aspect of the first embodiment, R^(2a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a seventeenth aspect of the first embodiment, R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In an eighteenth aspect of the first embodiment, R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a nineteenth aspect of the first embodiment, R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a twentieth aspect of the first embodiment, R⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a twenty-first aspect of the first embodiment, R^(7a) and R^(8a) are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above or in the first through fourteenth aspects described above.

In a twenty-second aspect of the first embodiment, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of —O—, —S—, —SO₂—, —CH₂—, and —CF₂—; X^(a) and X^(a1) are each independently selected from the group consisting of O and S; X^(b) and X^(b1) are each independently selected from the group consisting of O and S; X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each H; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆ haloalkyl, where said R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a) are each H; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position or optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above.

In a twenty-third aspect of the first embodiment, the compound of formula (I) is a compound of formula (Ia):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R³ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; and each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above.

In a twenty-fourth aspect of the first embodiment, the compound of formula (I) is a compound of formula (Ib):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R^(1a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R^(2a) is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R³ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) is selected from the group consisting of H, F, Cl, I, Br, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above.

In a twenty-fifth aspect of the first embodiment, the compound of formula (I) is a compound of formula (Ic):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R³ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R⁴ is selected from the group consisting of H, F, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) is selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, I, Br, and OH; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I) of the first embodiment above.

A twenty-sixth aspect of the first embodiment relates to a pharmaceutical composition, said pharmaceutical composition comprising (a) a compound according to any one of general formula (I) of the first embodiment above or in the first through twenty-fifth aspects described above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and (b) a pharmaceutically acceptable carrier.

A twenty-seventh aspect of the first embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) of the first embodiment above or in the first through twenty-fifth aspects described above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A twenty-eighth aspect of the first embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-sixth aspect described above to the subject.

A twenty-ninth aspect of the first embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) of the first embodiment above or in the first through twenty-fifth aspects described above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A thirtieth aspect of the first embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-sixth aspect described above to the subject.

A thirty-first aspect of the first embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) of the first embodiment above or in the first through twenty-fifth aspects described above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A thirty-second aspect of the first embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-sixth aspect described above to the subject.

A second embodiment of the disclosure relates to cyclic di-nucleotide compounds of general formula (I′):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃-6 cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of —O— and —S—; X^(a) and X^(a1) are each independently selected from the group consisting of O, and S; X^(b) and X^(b1) are each independently selected from the group consisting of O, and S; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(c1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R⁴ and R⁵ are connected to form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.

In specific aspects of this embodiment, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, F and OH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of,

either only one of R⁵ and R³ is selected from the group consisting of H, F, and OH, or neither R⁵ and R³ is selected from the group consisting of H, F, and OH. In further specific instances of this aspect, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆ alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² are each selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the second embodiment, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In particular instances, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In even more particular instances, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above.

In a second aspect of the second embodiment, X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹, where each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In particular instances, X^(c) and X^(c1) are each independently selected from the group consisting of O⁻, S⁻,

In all instances of this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first aspect described above.

In a third aspect of the second embodiment, R¹ and R^(1a) are each H. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through second aspects described above.

In a fourth aspect of the second embodiment, R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through third aspects described above.

In a fifth aspect of the second embodiment, R³ is selected from the group consisting H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R³ are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In even more particular instances, R³ is selected from NH₂ and N₃. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through fourth aspects described above.

In a sixth aspect of the second embodiment, R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particular instances, R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In even more particular instances, R⁴ and R^(4a) are each F. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through fifth aspects described above.

In a seventh aspect of the second embodiment, R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃. In particular instances, R⁵ are each independently selected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In even more particular instances, R⁵ is selected from NH₂ and N₃. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through sixth aspects described above.

In an eighth aspect of the second embodiment, R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, I, Br, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through seventh aspects described above.

In a ninth aspect of the second embodiment, R⁷ and R^(7a) are each independently selected from the group consisting of H and C₁-C₆ alkyl. In particular instances, R⁷ and R^(7a) are each independently selected from the group consisting of H and CH₃. In more particular instances, R^(7a) is CH₃. In additional instances, R⁷ and R^(7a) are each H. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eighth aspects described above.

In a tenth aspect of the second embodiment, R⁸ and R^(8a) are each independently selected from the group consisting of H and C₁-C₆ alkyl. In particular instances, R⁸ and R^(8a) are each independently selected from the group consisting of H and CH₃. In more particular instances, R^(8a) is CH₃. In additional instances, R⁸ and R^(8a) are each H. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through ninth aspects described above.

In an eleventh aspect of the second embodiment, R^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through tenth aspects described above.

In a twelfth aspect of the second embodiment, R^(2a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In a thirteenth aspect of the second embodiment, R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In a fourteenth aspect of the second embodiment, R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In a fifteenth aspect of the second embodiment, R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In a sixteenth aspect of the second embodiment, R⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In a seventeenth aspect of the second embodiment, R^(7a) and R^(8a) are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above or in the first through eleventh aspects described above.

In an eighteenth aspect of the second embodiment, Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of —O— and —S—; X^(a) and X^(a1) are each independently selected from the group consisting of O and S; X^(b) and X^(b1) are each independently selected from the group consisting of O and S; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each H; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆ haloalkyl, where said R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a) are each H; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position or optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In all instances of this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above.

In a nineteenth aspect of the second embodiment, the compound of formula (I′) is a compound of formula (I′a):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵ are not both selected from the group consisting of: OH, R⁵ C₁-C₆ alkyl substituted with OH, or C₁-C₆ haloalkyl substituted with OH; and each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In all instances of this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above.

In a twentieth aspect of the second embodiment, the compound of formula (I′) is a compound of formula (I′b):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R^(1a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above.

In a twenty-first aspect of the second embodiment, the compound of formula (I′) is a compound of formula (I′c):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect, all other groups are as provided in the general formula (I′) of the second embodiment above.

A twenty-second aspect of the second embodiment relates to a pharmaceutical composition, said pharmaceutically acceptable composition comprising (a) a compound according to any one of general formula (I′) of the second embodiment above or in the first through twenty-first aspects described above or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, and (b) a pharmaceutically acceptable carrier.

A twenty-third aspect of the second embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I′) of the second embodiment above or in the first through twenty-first aspects described above or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof to the subject.

A twenty-fourth aspect of the second embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-second aspect described above or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof to the subject.

A twenty-fifth aspect of the second embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general (I′) of the second embodiment above or in the first through twenty-first aspects described above or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof to the subject.

A twenty-sixth aspect of the second embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-second aspect described above to the subject.

A twenty-seventh aspect of the second embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general (I′) of the second embodiment above or in the first through twenty-first aspects described above or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof to the subject.

A thirty-third aspect of the second embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-second aspect described above to the subject.

A third embodiment of the disclosure relates to cyclic di-nucleotide compounds of general formula (I″):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.

In specific aspects of this embodiment, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, F and OH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

either only one of R⁵ and R³ is selected from the group consisting of H, F, and OH, or neither R⁵ and R³ is selected from the group consisting of H, F, and OH. In further specific instances of this aspect, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆ alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² are each selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the third embodiment, the compound of formula (I″) is a compound of formula (I″a):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; and each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In instances of this aspect, Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is F; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; and each R⁹ is independently H.

In a second aspect of the third embodiment, the compound of formula (I″) is a compound wherein R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.

In a third aspect of the third embodiment, the compound of formula (I″) is a compound of formula (I″b):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently H; and R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position. In instances of this aspect, Base¹ and Base² are each independently selected from the group consisting of

In a fourth aspect of the third embodiment, the compound of formula (I″) is a compound wherein at least one of Base¹ and Base² are each independently selected from the group consisting of

A fifth aspect of the third embodiment relates to a pharmaceutical composition, said pharmaceutical composition comprising (a) a compound according to any one of general formula (I″) of the third embodiment or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and (b) a pharmaceutically acceptable carrier.

A sixth aspect of the third embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to general formula (I″) of the third embodiment above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A seventh aspect of the third embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the fifth aspect described above to the subject.

An eighth aspect of the third embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according general formula (I″) of the third embodiment above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A ninth aspect of the third embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the fifth aspect described above to the subject.

A tenth aspect of the third embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to general formula (I″) of the third embodiment above or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject.

An eleventh aspect of the third embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the fifth aspect described above to the subject.

In an additional embodiment, the compound is selected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In aspects of this embodiment, the compound is selected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In particular aspects, the compound is selected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In more particular aspects of this embodiment, the compound is selected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In still more particular aspects, the compound is selected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

In another embodiment, for the compounds of general formula (I), compounds of general formula (I′) and compounds of general formula (I″), variables Base¹, Base², Y, Y^(a), X^(a), X^(a1), X^(b), X^(b1), X^(c), X^(c1), X^(d), X^(d1), R¹, R^(1a), R², R^(2a), R³, R⁴, R^(4a), R⁵, R⁶, R^(6a), R⁷, R^(7a), R⁸, R^(8a), R⁹, and R¹⁰ are each selected independently from each other.

In another embodiment of the disclosure, the compound of the disclosure is selected from the exemplary species depicted in Examples 1 through 348 shown below.

Other embodiments of the present disclosure include the following:

(a) A pharmaceutical composition comprising an effective amount of a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.

(b) The pharmaceutical composition of (a), further comprising a second therapeutic agent selected from the group consisting of STING agonist compounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents.

(c) A pharmaceutical combination that is (i) a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and (ii) a second therapeutic agent selected from the group consisting of STING agonist compounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents; wherein the a compound of general formula (I) or compound of general formula (I′), or compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and the second therapeutic agent are each employed in an amount that renders the combination effective for inducing an immune response in a patient.

(e) A method of inducing an immune response in a patient, which comprises administering to the subject an effective amount of a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

(f) A method of inducing an immune response in a patient, which comprises administering to the subject an effective amount of a composition of (a), a composition of (b) or a combination of (c).

(g) A method of inducing STING-dependent type I interferon production in a patient, which comprises administering to the subject an effective amount of (a), a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″).

(h) A method of inducing STING-dependent type I interferon production in a patient, which comprises administering to the subject an effective amount of a composition of (a), a composition of (b) or a combination of (c).

(i) A method of inducing STING-dependent cytokine production in a patient, which comprises administering to the subject an effective amount of a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

(j) A method of inducing STING-dependent cytokine production in a patient, which comprises administering to the subject an effective amount of a composition of (a), a composition of (b) or a combination of (c).

(k) A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a compound of general formula (I) or a compound of general formula (I′), or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to the subject;

(l) The method of (k), wherein the cell proliferation disorder is cancer.

(m). A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition of (a), a composition of (b) or a combination of (c) to the subject.

(n) The method of (m), wherein the cell proliferation disorder is cancer.

The present disclosure also includes a compound of the present disclosure for use (i) in, (ii) as a medicament for, or (iii) in the preparation of a medicament for: (a) inducing an immune response in a patient, or (b) inducing a STING-dependent cytokine production in a patient. In these uses, the compounds of the present disclosure can optionally be employed in combination with one or more second therapeutic agents selected from STING agonist compounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents.

Additional embodiments of the disclosure include the pharmaceutical compositions, combinations and methods set forth in (a)-(n) above and the uses set forth in the preceding paragraph, wherein the compound of the present disclosure employed therein is a compound of one of the embodiments, aspects, classes, sub-classes, or features of the compounds described above. In all of these embodiments, the compound may optionally be used in the form of a pharmaceutically acceptable salt, hydrate, solvate or prodrug as appropriate.

In the embodiments of the compound provided above, it is to be understood that each embodiment may be combined with one or more other embodiments, to the extent that such a combination provides a stable compound and is consistent with the description of the embodiments. It is further to be understood that the embodiments of compositions and methods provided as (a) through (n) above are understood to include all embodiments of the compounds, including such embodiments as result from combinations of embodiments.

The term “subject” (alternatively referred to herein as “patient”) as used herein refers to an animal, preferably a mammal, such as a human being, male or female, that has been the object of treatment, observation, or experiment. A subject also refers to one or more of cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, and birds. In embodiments, the subject is human.

As used herein, the term “immune response” relates to any one or more of the following: specific immune response, non-specific immune response, both specific and non-specific response, innate response, primary immune response, adaptive immunity, secondary immune response, memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. In certain embodiments, the compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, is administered in conjunction with one or more additional therapeutic agents including vaccines intended to stimulate an immune response to one or more predetermined anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents, etc. In certain embodiments, the compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, is administered in conjunction with one or more additional compositions including vaccines intended to stimulate an immune response to one or more predetermined anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents, etc.

Compounds

The term “alkyl” refers to a monovalent straight or branched chain, saturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range. Thus, for example, “C₁₋₆ alkyl” (or “C₁-C₆ alkyl”) refers to any of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and tert-butyl, n- and iso-propyl, ethyl, and methyl. As another example, “C₁₋₄ alkyl” refers to n-, iso-, sec- and tert-butyl, n- and isopropyl, ethyl, and methyl.

As used herein, the term “alkylene” refers to a bivalent straight chain, saturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range.

As used herein, the term “alkenyl” refers to a monovalent straight or branched chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more double bond.

As used herein, the term “alkenylene” refers to a bivalent straight chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more double bond.

As used herein, the term “alkynyl” refers to a monovalent straight or branched chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more triple bond.

As used herein, the term “alkynylene” refers to a bivalent straight chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more triple bond.

The term “halogen” (or “halo”) refers to fluorine, chlorine, bromine, and iodine (alternatively referred to as fluoro, chloro, bromo, and iodo or F, Cl, Br, and I).

The term “haloalkyl” refers to an alkyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen. Thus, for example, “C₁₋₆ haloalkyl” (or “C₁-C₆ haloalkyl”) refers to a C₁ to C₆ linear or branched alkyl group as defined above with one or more halogen substituents. The term “fluoroalkyl” has an analogous meaning except the halogen substituents are restricted to fluoro. Suitable fluoroalkyls include the series (CH₂)₀₋₄CF₃ (i.e., trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoro-n-propyl, etc.).

As used herein, the term “haloalkenyl” refers to an alkenyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen.

As used herein, the term “haloalkynyl” refers to an alkynyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen.

As used herein, the term “spirocycle” or “spirocyclic ring” refers to a pendant cyclic group formed by substituents on a single atom. For example, in general formula (I), a spirocycle may be formed by R^(2a) and R³.

Unless expressly stated to the contrary, all ranges cited herein are inclusive; i.e., the range includes the values for the upper and lower limits of the range as well as all values in between. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. Numerical values provided herein, and the use of the term “about”, may include variations of ±1%, ±2%, ±3%, ±4%, ±5%, ±10%, ±15%, and ±20% and their numerical equivalents.

As used herein, the term “one or more” item includes a single item selected from the list as well as mixtures of two or more items selected from the list.

In the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure is meant to include all suitable isotopic variations of the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″). For example, different isotopic forms of hydrogen (H) include protium (¹H) and deuterium (²H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds within general formula (I) can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.

In particular embodiments of the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), the compounds are isotopically enriched with deuterium. In aspects of these embodiments, one or more of R¹, R^(1a), R², R^(2a), R³, R⁴, R^(4a), R⁵, R⁶, R^(6a), R⁷, R^(7a), R⁸, R^(8a), R⁹, and R¹⁰ may be deuterium.

As shown in the general structural formulas and the structures of specific compounds as provided herein, a straight line at a chiral center includes both (R) and (S) stereoisomers and mixtures thereof. Also, unless otherwise specified (e.g., 100% purified compound), reference to a particular stereochemistry at a position provides a compound having the indicated stereochemistry, but does not exclude the presence of stereoisomers having different stereochemistry at the indicated position.

Recitation or depiction of a specific compound in the claims (i.e., a species) without a specific stereoconfiguration designation, or with such a designation for less than all chiral centers, is intended to encompass the racemate, racemic mixtures, each individual enantiomer, a diastereoisomeric mixture and each individual diastereomer of the compound where such forms are possible due to the presence of one or more asymmetric centers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of general formula (I), a compound of general formula (I′), and/or a compound of general formula (I″), or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates, which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Alternatively, absolute stereochemistry may be determined by Vibrational Circular Dichroism (VCD) spectroscopy analysis. The present invention includes all such isomers, as well as salts, solvates (which includes hydrates) and solvated salts of such racemates, enantiomers, diastereomers and tautomers and mixtures thereof.

Those skilled in the art will recognize that chiral compounds, and in particular sugars, can be drawn in a number of different ways that are equivalent. Those skilled in the art will further recognize that the identity and regiochemical position of the substituents on ribose can vary widely and that the same principles of stereochemical equivalence apply regardless of substituent. Non-limiting examples of such equivalence include those exemplified below.

Salts

Compounds described herein having appropriate functional groups can be provided as salts. Examples of such compounds are described herein by reference to possible salts. Such reference is for illustration only. Additional embodiments include salts of any compounds described herein having suitable groups.

Pharmaceutically acceptable salts can be used with compounds for treating patients. Non-pharmaceutical salts may, however, be useful in the preparation of intermediate compounds.

Pharmaceutically acceptable salts are suitable for administration to a patient, preferably, a human. Suitable salts include acid addition salts that may, for example, be formed by mixing a solution of a compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. Compounds carrying an acidic moiety can be mixed with suitable pharmaceutically acceptable salts to provide, for example, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (—CO0H) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.

Methods of Preparing Compounds

Several methods for preparing the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, are described in the following Schemes and Examples. Starting materials and intermediates are purchased from commercial sources, made from known procedures, or are otherwise illustrated. In some cases the order of carrying out the steps of the reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.

Method 1

One method for the preparation of examples of the disclosure is detailed in Scheme 1. This procedure was adequately modified from the previously reported procedure for cyclic dinucleotide synthesis (Barbara L. Gaffney et al., One-Flask Syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] Thiophosphate Analogues, 12 ORG. LETT. 3269-3271 (2010)). The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, a phosphoramidite functionality at 2′-O position, and DMTr ether at 5′-O position. It was treated with aqueous TFA/pyridine condition and subsequently t-butylamine to convert the 2′-phosphoramidite moiety to an H-phosphonate. Then, DMTr ether was removed under acidic condition. The resulting 5′-hydroxyl group was reacted with 3′-phosphoramidites of fully protected second modified ribo-nucleoside to give a cyclized compound. It was immediately oxidized with t-butyl hydroperoxide. Then, the 5′-hydroxyl group of the second ribo-nucleoside was deprotected with dichloroacetic acid. Using 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide as a coupling reagent, the H-phosphonate at 2′-O of the first ribo-nucleoside was reacted with 5′-OH of the second ribo-nucleoside to give a cyclic product. It was immediately oxidized with aqueous iodine. Treatment with t-butylamine and methylamine plus fluoride anion in case silyl protection was used provided the desired cyclic dinucleotide 1G.

Method 2

Another method for the preparation of examples of the disclosure is detailed in Scheme 2. This procedure was modified from Scheme 1. The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, a phosphoramidite functionality at 2′-O position, and DMTr ether at 5′-O position. It was treated with aqueous TFA/pyridine condition and subsequently t-butylamine to convert the 2′-phosphoramidite moiety to an H-phosphonate. Then, DMTr ether was removed under acidic condition. The resulting 5′-hydroxyl group was reacted with 3′-phosphoramidites of fully protected second modified ribo-nucleoside to give a cyclized compound. It was immediately thioated with (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide. Then, the 5′-hydroxyl group of the second ribo-nucleoside was deprotected with dichloroacetic acid. Using 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide as a coupling reagent, the H-phosphonate at 2′-O of the first ribo-nucleoside was reacted with 5′-OH of the second ribo-nucleoside to give a cyclic product. It was immediately thioated with 3H-benzo[c][1,2]dithiol-3-one. Treatment with t-butylamine and methylamine plus fluoride anion in case silyl protection was used provided the desired cyclic dinucleotide diphosphorothioate 2G.

Methods of Use

Compounds described herein having therapeutic applications, such as the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), and the compounds of the Examples 1 through 348, can be administered to a patient for the purpose of inducing an immune response, inducing a STING-dependent cytokine production and/or inducing anti-tumor activity. The term “administration” and variants thereof (e.g., “administering” a compound) means providing the compound to the individual in need of treatment. When a compound is provided in combination with one or more other active agents (e.g., antiviral agents useful for treating HCV infection or anti-tumor agents for treating cancers), “administration” and its variants are each understood to include concurrent and sequential provision of the compound or salt and other agents.

The compounds disclosed herein are STING agonists and inhibitors of viral replication. These compounds are potentially useful in treating diseases or disorders including, but not limited to, cell proliferation disorders, such as cancer.

Cell-proliferation disorders include, but are not limited to, cancer. Examples of such cancers include, but are not limited to, Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Adrenocortical Carcinoma; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma; Bile Duct Cancer; Bladder Cancer; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor, Medulloblastoma; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; Breast Cancer; Bronchial Adenomas/Carcinoids; Carcinoid Tumor; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Central Nervous System Lymphoma, Primary; Cerebral Astrocytoma/Malignant Glioma; Cervical Cancer; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer; Ewing's Family of Tumors; Extracranial Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer; Hodgkin's Lymphoma; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer; Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin's Lymphoma; Non-Small Cell Lung Cancer; Oral Cancer; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; steosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, Soft Tissue; Sezary Syndrome; Skin Cancer; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Supratentorial Primitive Neuroectodermal Tumors; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Malignant; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

In one embodiment, the cancer is brain cancer, such as an astrocytic tumor (e.g., pilocytic astrocytoma, subependymal giant-cell astrocytoma, diffuse astrocytoma, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, astrocytoma, giant cell glioblastoma, glioblastoma, secondary glioblastoma, primary adult glioblastoma, and primary pediatric glioblastoma); oligodendroglial tumor (e.g., oligodendroglioma, and anaplastic oligodendroglioma); oligoastrocytic tumor (e.g., oligoastrocytoma, and anaplastic oligoastrocytoma); ependymoma (e.g., myxopapillary ependymoma, and anaplastic ependymoma); medulloblastoma; primitive neuroectodermal tumor, schwannoma, meningioma, atypical meningioma, anaplastic meningioma; and pituitary adenoma. In another embodiment, the brain cancer is glioma, glioblastoma multiforme, paraganglioma, or suprantentorial primordial neuroectodermal tumors (sPNET).

In another embodiment, the cancer is leukemia, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), myeloproliferative neoplasm (MPN), post-MPN AML, post-MDS AML, del(5q)-associated high risk MDS or AML, blast-phase chronic myelogenous leukemia, angioimmunoblastic lymphoma, and acute lymphoblastic leukemia.

In one embodiment, the cancer is skin cancer, including melanoma. In another embodiment, the cancer is prostate cancer, breast cancer, thyroid cancer, colon cancer, or lung cancer. In another embodiment, the cancer is sarcoma, including central chondrosarcoma, central and periosteal chondroma, and fibrosarcoma. In another embodiment, the cancer is cholangiocarcinoma.

As used herein, the terms “treatment” and “treating” refer to all processes wherein there may be a slowing, interrupting, arresting, controlling, or stopping of the progression of a disease or disorder described herein. The terms do not necessarily indicate a total elimination of all disease or disorder symptoms.

The terms “administration of” and or “administering” a compound should be understood to include providing a compound described herein, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, and compositions of the foregoing to a subject.

The amount of a compound administered to a subject is an amount sufficient to induce an immune response and/or to induce STING-dependent type I interferon production in the subject. In an embodiment, the amount of a compound can be an “effective amount” or “therapeutically effective amount,” wherein the subject compound is administered in an amount that will elicit, respectively, a biological or medical (i.e., intended to treat) response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. An effective amount does not necessarily include considerations of toxicity and safety related to the administration of a compound.

An effective amount of a compound will vary with the particular compound chosen (e.g., considering the potency, efficacy, and/or half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the subject being treated; the medical history of the subject being treated; the duration of the treatment; the nature of a concurrent therapy; the desired therapeutic effect; and like factors and can be routinely determined by the skilled artisan.

The compounds disclosed herein may be administered by any suitable route including oral and parenteral administration. Parenteral administration is typically by injection or infusion and includes intravenous, intramuscular, and subcutaneous injection or infusion.

The compounds disclosed herein may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound disclosed herein depend on the pharmacokinetic properties of that compound, such as absorption, distribution and half-life which can be determined by a skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for a compound disclosed herein depend on the disease or condition being treated, the severity of the disease or condition, the age and physical condition of the subject being treated, the medical history of the subject being treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual subject's response to the dosing regimen or over time as the individual subject needs change. Typical daily dosages may vary depending upon the particular route of administration chosen.

One embodiment of the present disclosure provides for a method of treating a cell proliferation disorder comprising administration of a therapeutically effective amount of a compound of general formula (I), a compound of general formula (I′), or a compound of general formula (I″) to a subject in need of treatment thereof. In one embodiment, the cell proliferation disorder is cancer.

In one embodiment, the cancer is brain cancer, leukemia, skin cancer, prostate cancer, thyroid cancer, colon cancer, lung cancer or sarcoma. In another embodiment the cancer is selected from the group consisting of glioma, glioblastoma multiforme, paraganglioma, suprantentorial primordial neuroectodermal tumors, acute myeloid leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, melanoma, breast, prostate, thyroid, colon, lung, central chondrosarcoma, central and periosteal chondroma tumors, fibrosarcoma, and cholangiocarcinoma.

In one embodiment, disclosed herein is the use of a compound of general formula (I), compound of general formula (I′), and/or compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, in a therapy. The compound may be useful in a method of inducing an immune response and/or inducing STING-dependent type I interferon production in a subject, such as a mammal in need of such inhibition, comprising administering an effective amount of the compound to the subject.

In one embodiment, disclosed herein is a pharmaceutical composition comprising at least one compound of general formula (I), compound of general formula (I′), and/or compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, for use in potential treatment to induce an immune response and/or to induce STING-dependent type I interferon production.

In one embodiment, disclosed herein is the use of a compound of general formula (I), compound of general formula (I′), and/or compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, in the manufacture of a medicament for the treatment to induce an immune response and/or to induce STING-dependent type I interferon production. In one embodiment, the disease or disorder to be treated is a cell proliferation disorder. In another embodiment, the cell proliferation disorder is cancer. In another embodiment, the cancer is brain cancer, leukemia, skin cancer, breast, prostate cancer, thyroid cancer, colon cancer, lung cancer, or sarcoma. In another embodiment, the cancer is glioma, glioblastoma multiforme, paraganglioma, suprantentorial primordial neuroectodermal tumors, acute myeloid leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, melanoma, breast, prostate, thyroid, colon, lung, central chondrosarcoma, central and periosteal chondroma tumors, fibrosarcoma, and/or cholangiocarcinoma.

Compositions

The term “composition” as used herein is intended to encompass a dosage form comprising a specified compound in a specified amount, as well as any dosage form which results, directly or indirectly, from combination of a specified compound in a specified amount.

Such term is intended to encompass a dosage form comprising a compound of general formula (I), a compound of general formula (I′), and/or a compound of general formula (I″), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients. Accordingly, the compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and one or more pharmaceutically acceptable carrier or excipients. By “pharmaceutically acceptable”, it is meant the carriers or excipients are compatible with the compound disclosed herein and with other ingredients of the composition.

For the purpose of inducing an immune response and/or inducing a STING-dependent type I interferon production, the compounds, optionally in the form of a salt, hydrate, solvate or prodrug, can be administered by means that produces contact of the active agent with the agent's site of action. They can be administered by conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but typically are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

In one embodiment, disclosed herein is a composition comprising a compound of general formula (I), a compound of general formula (I′), and/or a compound of general formula (I″), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients. The composition may be prepared and packaged in bulk form wherein an effective amount of a compound of the disclosure can be extracted and then given to a subject, such as with powders or syrups. Alternatively, the composition may be prepared and packaged in unit dosage form wherein each physically discrete unit contains an effective amount of a compound of general formula (I), a compound of general formula (I′), and/or a compound of general formula (I″).

The compounds disclosed herein and a pharmaceutically acceptable carrier or excipient(s) will typically be formulated into a dosage form adapted for administration to a subject by a desired route of administration. For example, dosage forms include those adapted for (1) oral administration, such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; and (2) parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution. Suitable pharmaceutically acceptable carriers or excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable carriers or excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the carrying or transporting of a compound disclosed herein, once administered to the subject, from one organ or portion of the body to another organ or another portion of the body. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, lubricants, binders, disintegrants, fillers, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anti-caking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.

A skilled artisan possesses the knowledge and skill in the art to select suitable pharmaceutically acceptable carriers and excipients in appropriate amounts for the use in the disclosure. In addition, there are a number of resources available to the skilled artisan, which describe pharmaceutically acceptable carriers and excipients and may be useful in selecting suitable pharmaceutically acceptable carriers and excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

The compositions of the disclosure are prepared using techniques and methods known to those skilled in the art. Some methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

In one embodiment, the disclosure is directed to a solid oral dosage form such as a tablet or capsule comprising an effective amount of a compound of the disclosure and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives, (e.g., microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pre-gelatinized starch) gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The composition can also be prepared to prolong or sustain the release as, for example, by coating or embedding particulate material in polymers, wax, or the like.

The compounds disclosed herein may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the disclosure may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanacrylates and cross-linked or amphipathic block copolymers of hydrogels.

In one embodiment, the disclosure is directed to a liquid oral dosage form. Oral liquids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of a compound disclosed herein. Syrups can be prepared by dissolving the compound of the disclosure in a suitably flavored aqueous solution; while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing a compound disclosed herein in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additives such as peppermint oil or other natural sweeteners or saccharin or other artificial sweeteners and the like can also be added.

In one embodiment, the disclosure is directed to compositions for parenteral administration. Compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Combinations

The compounds of general formula (I), compounds of general formula (I′), and/or compounds of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, may be administered in combination with one or more additional therapeutic agents. In embodiments, one or more a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and the one or more additional therapeutic agents may be co-adminstered. The additional therapeutic agent(s) may be administered in a single dosage form with the compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, or the additional therapeutic agent(s) may be administered in separate dosage form(s) from the dosage form containing the compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. The additional therapeutic agent(s) may be one or more agents selected from the group consisting of STING agonist compounds, anti-viral compounds, antigens, adjuvants, anti-cancer agents, CTLA-4, LAG-3 and PD-1 pathway antagonists, lipids, peptides, cytotoxic agents, chemotherapeutic agents, immunomodulatory cell lines, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, topoisomerase II inhibitors, smoothen inhibitors, alkylating agents, anti-tumor antibiotics, anti-metabolites, retinoids, and immunomodulatory agents including but not limited to anti-cancer vaccines. It will be understood the descriptions of the above additional therapeutic agents may be overlapping. It will also be understood that the treatment combinations are subject to optimization, and it is understood that the best combination to use of the compounds of general formula (I), compounds of general formula (I′), or compounds of general formula (I″) and one or more additional therapeutic agents will be determined based on the individual patient needs.

A compound disclosed herein may be used in combination with one or more other active agents, including but not limited to, other anti-cancer agents that are used in the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition (e.g., cell proliferation disorders). In one embodiment, a compound disclosed herein is combined with one or more other anti-cancer agents for use in the prevention, treatment, control amelioration, or reduction of risk of a particular disease or condition for which the compounds disclosed herein are useful. Such other active agents may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure.

When a compound disclosed herein is used contemporaneously with one or more other active agents, a composition containing such other active agents in addition to the compound disclosed herein is contemplated. Accordingly, the compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to a compound disclosed herein. A compound disclosed herein may be administered either simultaneously with, or before or after, one or more other therapeutic agent(s). A compound disclosed herein may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agent(s).

Products provided as combinations may include a composition comprising a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and one or more other active agent(s) together in the same pharmaceutical composition, or may include a composition comprising a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a composition comprising one or more other therapeutic agent(s) in separate form, e.g. in the form of a kit or in any form designed to enable separate administration either concurrently or on separate dosing schedules.

The weight ratio of a compound disclosed herein to a second active agent may be varied and will depend upon the effective dose of each agent. Generally, an effective dose of each will be used. Combinations of a compound disclosed herein and other active agents will generally also be within the aforementioned range, but in each case, an effective dose of each active agent should be used. In such combinations, the compound disclosed herein and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

In one embodiment, this disclosure provides a composition comprising a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a cell proliferation disorder, such as cancer.

In one embodiment, the disclosure provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules, and the like.

A kit of this disclosure may be used for administration of different dosage forms, for example, oral and parenteral, for administration of the separate compositions at different dosage intervals, or for titration of the separate compositions against one another. To assist with compliance, a kit of the disclosure typically comprises directions for administration.

Disclosed herein is a use of a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, for treating a cell proliferation disorder, wherein the medicament is prepared for administration with another active agent. The disclosure also provides the use of another active agent for treating a cell proliferation disorder, wherein the medicament is administered with a compound of general formula (I).

The disclosure also provides the use of a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, for treating a cell proliferation disorder, wherein the patient has previously (e.g., within 24 hours) been treated with another active agent. The disclosure also provides the use of another therapeutic agent for treating a cell proliferation disorder, wherein the patient has previously (e.g., within 24 hours) been treated with a compound of general formula (I), compound of general formula (I′), or compound of general formula (I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. The second agent may be applied a week, several weeks, a month, or several months after the administration of a compound disclosed herein.

STING agonist compounds that may be used in combination with the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″) disclosed herein include but are not limited to cyclic di-nucleotide compounds.

Anti-viral compounds that may be used in combination with the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″) disclosed herein include hepatitis B virus (HBV) inhibitors, hepatitis C virus (HCV) protease inhibitors, HCV polymerase inhibitors, HCV NS4 Å inhibitors, HCV NS5 Å inhibitors, HCV NS5b inhibitors, and human immunodeficiency virus (HIV) inhibitors.

Antigens and adjuvants that may be used in combination with the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″) disclosed herein include B7 costimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40 ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. Adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, a toll-like receptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, LPS, monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition retinoic acid-inducible gene I (RIG-I) agonists such as poly I:C, used separately or in combination with the described compositions are also potential adjuvants.

CLTA-4 and PD-1 pathways are important negative regulators of immune response. Activated T-cells upregulate CTLA-4, which binds on antigen-presenting cells and inhibits T-cell stimulation, IL-2 gene expression, and T-cell proliferation; these anti-tumor effects have been observed in mouse models of colon carcinoma, metastatic prostate cancer, and metastatic melanoma. PD-1 binds to active T-cells and suppresses T-cell activation; PD-1 antagonists have demonstrated anti-tumor effects as well. CTLA-4 and PD-1 pathway antagonists that may be used in combination with the compounds of general formula (I) disclosed herein include ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011, AMP-224, and MDX-1106.

“PD-1 antagonist” or “PD-1 pathway antagonist” means any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T-cell, B-cell, or NKT-cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1 L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1; and PDCD1 L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of the treatment method, medicaments and uses of the present disclosure in which a human individual is being treated, the PD-1 antagonist blocks binding of human PD-L1 to human PD-1, and preferably blocks binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in any of the treatment method, medicaments and uses of the present disclosure include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab′-SH, F(ab′)₂, scFv, and Fv fragments.

Examples of mAbs that bind to human PD-1, and useful in the treatment method, medicaments and uses of the present disclosure, are described in U.S. Pat. No. 7,488,802, U.S. Pat. No. 7,521,051, U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,354,509, and U.S. Pat. No. 8,168,757, PCT International Patent Application Publication Nos. WO2004/004771, WO2004/072286, and WO2004/056875, and U.S. Patent Application Publication No. US2011/0271358.

Examples of mAbs that bind to human PD-L1, and useful in the treatment method, medicaments and uses of the present disclosure, are described in PCT International Patent Application Nos. WO2013/019906 and WO2010/077634 A1 and in U.S. Pat. No. 8,383,796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present disclosure include MPDL3280 Å, BMS-936559, MEDI4736, MSB0010718C, and an antibody that comprises the heavy chain and light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21, respectively, of WO2013/019906.

Other PD-1 antagonists useful in any of the treatment method, medicaments, and uses of the present disclosure include an immune-adhesion that specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1, e.g., a fusion protein containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immune-adhesion molecules that specifically bind to PD-1 are described in PCT International Patent Application Publication Nos. WO2010/027827 and WO2011/066342. Specific fusion proteins useful as the PD-1 antagonist in the treatment method, medicaments, and uses of the present disclosure include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.

Examples of cytotoxic agents that may be used in combination with the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, disclosed herein include, but are not limited to, arsenic trioxide (sold under the tradename TRISENOX®), asparaginase (also known as L-asparaginase, and Erwinia L-asparaginase, sold under the tradenames ELSPAR® and KIDROLASE®).

Chemotherapeutic agents that may be used in combination with the compounds of general formula (I), compounds of general formula (I′), and compounds of general formula (I″), or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, disclosed herein include abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-tbutylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3′,4′-didehydro-4′deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, nivolumab, onapristone, paclitaxel, pembrolizumab, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine.

Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include, but are not limited to, bevacizumab (sold under the trademark AVASTIN by Genentech/Roche), axitinib (described in PCT International Patent Publication No. WO1/002369), Brivanib Alaninate ((S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate, also known as BMS-582664), motesanib (N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide. and described in PCT International Patent Application Publication No. WO02/068470), pasireotide (also known as SO 230, and described in PCT International Patent Publication No. WO02/010192), and sorafenib (sold under the tradename NEXAVAR).

Examples of topoisomerase II inhibitors, include but are not limited to, etoposide (also known as VP-16 and Etoposide phosphate, sold under the tradenames TOPOSAR, VEPESID, and ETOPOPHOS), and teniposide (also known as VM-26, sold under the tradename VUMON).

Examples of alkylating agents, include but are not limited to, 5-azacytidine (sold under the trade name VIDAZA), decitabine (sold under the trade name of DECOGEN), temozolomide (sold under the trade names TEMODAR and TEMODAL by Schering-Plough/Merck), dactinomycin (also known as actinomycin-D and sold under the tradename COSMEGEN), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under the tradename ALKERAN), altretamine (also known as hexamethylmelamine (HMM), sold under the tradename HEXALEN), carmustine (sold under the tradename BCNU), bendamustine (sold under the tradename TREANDA), busulfan (sold under the tradenames BUSULFEX® and MYLERAN®), carboplatin (sold under the tradename PARAPLATIN®), lomustine (also known as CCNU, sold under the tradename CEENU®), cisplatin (also known as CDDP, sold under the tradenames PLATINOL® and PLATINOL®-AQ), chlorambucil (sold under the tradename LEUKERAN®), cyclophosphamide (sold under the tradenames CYTOXAN® and NEOSAR®), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, sold under the tradename DTIC-DOME®), altretamine (also known as hexamethylmelamine (HMM) sold under the tradename HEXALEN®), ifosfamide (sold under the tradename IFEX®), procarbazine (sold under the tradename MATULANE®), mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, sold under the tradename MUSTARGEN®), streptozocin (sold under the tradename ZANOSAR®), thiotepa (also known as thiophosphoamide, TESPA and TSPA, and sold under the tradename THIOPLEX®.

Examples of anti-tumor antibiotics include, but are not limited to, doxorubicin (sold under the tradenames ADRIAMYCIN® and RUBEX®), bleomycin (sold under the tradename LENOXANE®), daunorubicin (also known as dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, sold under the tradename CERUBIDINE®), daunorubicin liposomal (daunorubicin citrate liposome, sold under the tradename DAUNOXOME®), mitoxantrone (also known as DHAD, sold under the tradename NOVANTRONE®), epirubicin (sold under the tradename ELLENCE™), idarubicin (sold under the tradenames IDAMYCIN®, IDAMYCIN PFS®), and mitomycin C (sold under the tradename MUTAMYCIN®).

Examples of anti-metabolites include, but are not limited to, claribine (2-chlorodeoxyadenosine, sold under the tradename LEUSTATIN®), 5-fluorouracil (sold under the tradename ADRUCIL®), 6-thioguanine (sold under the tradename PURINETHOL®), pemetrexed (sold under the tradename ALIMTA®), cytarabine (also known as arabinosylcytosine (Ara-C), sold under the tradename CYTOSAR-U®), cytarabine liposomal (also known as Liposomal Ara-C, sold under the tradename DEPOCYT™), decitabine (sold under the tradename DACOGEN®), hydroxyurea (sold under the tradenames HYDREA®, DROXIA™ and MYLOCEL™), fludarabine (sold under the tradename FLUDARA®), floxuridine (sold under the tradename FUDR®), cladribine (also known as 2-chlorodeoxyadenosine (2-CdA) sold under the tradename LEUSTATIN™), methotrexate (also known as amethopterin, methotrexate sodium (MTX), sold under the tradenames RHEUMATREX® and TREXALL™), and pentostatin (sold under the tradename NIPENT®).

Examples of retinoids include, but are not limited to, alitretinoin (sold under the tradename PANRETIN®), tretinoin (all-trans retinoic acid, also known as ATRA, sold under the tradename VESANOID®), Isotretinoin (13-c/s-retinoic acid, sold under the tradenames ACCUTANE®, AMNESTEEM®, CLARAVIS®, CLARUS®, DECUTAN®, ISOTANE®, IZOTECH®, ORATANE®, ISOTRET®, and SOTRET®), and bexarotene (sold under the tradename TARGRETIN®).

Activity: STING Biochemical [3H]cGAMP Competition Assay

The individual compounds described in the Examples herein are defined as STING agonists by demonstrating binding to the STING protein with an EC₅₀ of 20 uM or less in the STING Biochemical [3H]cGAMP Competition Assay and demonstrating interferon production with a 20% or greater luminescence induction at 30 uM in the IFN-β secretion in the THP1 cell assay.

The ability of compounds to bind STING is quantified by the ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Hi-Five cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.

Abbreviations

-   ¹H-NMR Proton nuclear magnetic resonance spectroscopy -   ¹⁹F-NMR ¹⁹F nuclear magnetic resonance spectroscopy -   ³¹P-NMR ³¹P nuclear magnetic resonance spectroscopy -   Å Angstrom -   A^(Bz) 6-N-benzoyladenine -   aq Aqueous -   Ar Argon -   ATP Adenosine 5′-triphosphate -   Bz Benzoyl -   CD₃OD Deuterium-enriched methyl alcohol, deuterium-enriched methanol -   CHCl₃ Trichloromethane -   Ci Curie, a non-standard unit of radioactivity; 1Ci=3.7×10¹⁰Bq,     where Bq is Becquerel, the SI unit of radioactivity, equivalent to 1     disintegration per second (dps) -   CO₂ Carbon dioxide -   d Doublet -   d Day(s) -   D₂O Deuterium-enriched water -   DCA Dichloroacetic acid -   DCM, CH₂Cl₂ Dichloromethane -   ddd Doublet of doublet of doublet -   ddt Doublet of doublet of triplet -   DDTT     (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide -   DMF N,N-dimethylformamide -   DMOCP 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphineane 2-oxide -   DMSO Dimethyl sulfoxide -   DMTr 4,4′-dimethoxytrityl -   DMTrCl 4,4′-dimethoxytrityl chloride -   dq Doublet of quartet -   EC₅₀ half maximal effective concentration, concentration of a drug,     antibody or toxicant that induces a response halfway between the     baseline and maximum after a specified exposure time -   eq Equivalents -   ES Electron spray -   Et Ethyl -   Et₂O Diethyl ether -   Et₃ SiH Triethylsilane -   EtOAc Ethyl acetate -   EtOH Ethyl alcohol, ethanol -   g Gram -   GTP Guanosine 5′-triphosphate -   h Hour -   H₂O Water -   HEPES 2-[4-(2-hydroxyethyl)piperazin 1-yl]ethanesulfonic acid, a     zwitterionic organic chemical buffering agent -   hept Heptet -   Hex Hexanes -   HF-Pyr Hydrogen fluoride-pyridine complex -   HPLC High performance liquid chromatography -   Hz Hertz -   ITP Inosine 5′-triphosphate -   J NMR Coupling constant -   LCMS Liquid chromatography-mass spectroscopy -   m Multiplet -   M Molar, moles per liter -   mCi Millicurie -   Me Methyl -   MeCN Acetonitrile -   MeNH₂ Methylamine -   mg Milligram -   MgCl₂ Magnesium chloride -   MHz Megahertz -   min Minute(s) -   mL, ml Milliliter -   mM Millimole per liter -   mmol Millimole -   MOI Multiplicity of infection -   MPLC Medium pressure liquid chromatography -   MTBE Methyl t-butyl ether, methyl tertiary butyl ether -   Na₂SO₄ Sodium sulfate -   NaCl Sodium chloride -   NaHCO₃ Sodium bicarbonate -   NaHSO₃ Sodium bisulfite -   NaOH Sodium hydroxide -   ng Nanogram(s) -   NH₄HCO₃ Ammonium bicarbonate -   NH₄OH Ammonium hydroxide -   nL Nanoliter -   nm Nanometer -   nM Nanomolar -   P₂O₅ Phosphorus pentoxide -   Py Pyridine -   q Quartet -   RPM, rpm Revolutions per minute -   RT, rt Room temperature, approximately 25° C. -   s Singlet -   sat Saturated -   t Triplet -   TB S t-Butyldimethylsilyl -   TMA Trimethylamine -   TEA, Et₃N Triethylamine -   TFA Trifluoroacetic acid -   TLC Thin layer chromatography -   TMSCl Trimethylsilyl chloride -   T_(R) Retention time -   TrisCl Tris(hydroxymethyl)aminomethane hydrochloride -   v/v Volume/volume -   λ_(em) Emission wavelength -   λ_(ex) Excitation wavelength -   μg Microgram -   μL, uL Microliter -   μM, uM Micromolar

Preparation 1: N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

Step 1: (2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-3-yl benzoate

To a mixture of 3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (2.36 g, 17.4 mmol) in NMP (50 ml) was added NaH (60%, 0.744 g, 18.6 mmol). The mixture was vigorously stirred and after 1h, generation of bubbles had completely ceased. The mixture was added to ((2R,3R,4S,5R)-3-(benzoyloxy)-5-bromo-4-fluorotetrahydrofuran-2-yl)methyl benzoate (neat, 5.25 g, 12.4 mmol) in one portion. The reaction was stirred for 18h. LCMS showed several peaks with the desired mass (m/e=479). EtOAc (70 mL) and water (70 mL) were added to the reaction. Layers were separated, and the organic layer was washed with half saturated brine (3×10 mL) and brine (1×10 mL), dried (MgSO₄), and concentrated. The crude was purified via silica column eluting with 0 to 50% EtOAc in Hex to give the product. LCMS (ES, m/z): 479.3 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 8.62 (s, 1H), 8.34 (s, 1H), 8.28 (s, 1H), 8.10-8.04 (m, 2H), 7.97-7.90 (m, 2H), 7.77-7.69 (m, 1H), 7.67-7.55 (m, 3H), 7.49-7.42 (m, 2H), 6.97 (dd, J=6.5, 3.1 Hz, 1H), 6.49 (dt, J=17.6, 6.9 Hz, 1H), 6.16 (dt, J=56, 6.6 Hz, 1H), 4.76-4.62 (m, 3H).

Step 2. (2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of (2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-3-yl benzoate (2.00 g, 4.18 mmol) in pyridine (10 mL) at was added NH₃ in MeOH (7N, 20 mL, 140 mmol). It was stirred for 48h. LCMS showed completion of the reaction (m/e=271). It was concentrated and purified by silica column chromatography eluting with 10% MeOH in CH₂Cl₂ to give the desired product. LCMS (ES, m/z): 271.1 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d6): δ 8.54 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H), 6.73 (dd, J=6.5, 2.6 Hz, 1H), 6.00 (d, J=5.4 Hz, 1H), 5.51 (ddd, J=53, 7.2, 6.5 Hz, 1H), 4.93 (t, J=5.8 Hz, 1H), 4.86-4.74 (m, 1H), 3.91-3.83 (m, 1H), 3.77-3.61 (m, 2H).

Step 3: N-(3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of (2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (1.34 g, 4.96 mmol) in pyridine (30 mL) at 0° C. was added TMSCl (1.46 mL, 11.4 mmol). It was warmed to rt and stirred for 1h. Then, it was recooled to 0° C. and BzCl (0.921 mL, 7.93 mmol) was added dropwise. The reaction was slowly warmed to rt over 2h. LCMS showed completion of reaction (m/e=375, 479). Water (3 mL) was added. It was cooled to 0° C. and NH₃ in MeOH (7N, 2.8 mL, 20 mmol) was added. After 1h, the reaction mixture was concentrated. It was purified by silica column chromatography eluting with 0 to 10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 375.2 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 11.95 (s, 1H), 8.98 (s, 1H), 8.10 (d, J=7.6 Hz, 2H), 7.73-7.66 (m, 1H), 7.59 (t, J=7.7 Hz, 2H), 6.91 (d, J=6.2 Hz, 1H), 6.06 (d, J=5.6 Hz, 1H), 5.59 (t, J=53, 6.8 Hz, 1H), 4.90 (t, J=5.8 Hz, 1H), 4.82 (dq, J=19.8, 7.0 Hz, 1H), 3.92 (td, J=7.6, 2.9 Hz, 1H), 3.75 (ddd, J=12.1, 5.6, 3.0 Hz, 1H), 3.66 (dt, J=12.0, 6.6 Hz, 1H).

Step 4: N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of N-(3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide (1.25 g, 3.34 mmol) in pyridine (15 mL) at 0° C. was added DMTrCl (1.58 g, 4.68 mmol). It was stirred at rt for 1h. LCMS showed a peak with the desired mass (m/e=677). It was partly concentrated (to 5 mL), and EtOAc (20 mL) and water (10 mL) were added. Layers were separated, and the aq layer was extracted with EtOAc (2×10 mL). The combined organics were washed with brine (5 mL), dried (MgSO₄), concentrated and purified by silica column chromatography eluting with 0 to 60% EtOAc in Hex to give the product. LCMS (ES, m/z): 675.5 [M−H]⁻. ¹H-NMR (500 MHz, DMSO-d₆): δ 8.13-8.07 (m, 2H), 7.69 (t, J=7.4 Hz, 1H), 7.59 (t, J=7.6 Hz, 2H), 7.35-7.29 (m, 2H), 7.23-7.10 (m, 6H), 6.97 (d, J=6.5 Hz, 1H), 6.81-6.74 (m, 2H), 6.74-6.67 (m, 2H), 6.07 (d, J=5.7 Hz, 1H), 5.62 (dt, J=53, 7.0 Hz, 1H), 4.91-4.79 (m, 1H), 4.15-4.07 (m, 1H), 3.69 (s, 3H), 3.67 (s, 3H), 3.44 (dd, J=10.4, 8.0 Hz, 1H), 3.21 (dd, J=10.3, 2.4 Hz, 1H).

Preparation 2: (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

Step 1: (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

To a solution of 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (8.02 g, 26.6 mmol) in ACN (90 mL) at rt was added pyridin-1-ium 2,2,2-trifluoroacetate (3.85 g, 19.95 mmol) and a solution of N-(3-((2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide (9 g, 13.30 mmol) in ACN (90 mL). The resulting mixture was stirred for 1h. Then, it was concentrated and the residue was dissolved in CH₂Cl₂ (1000 mL). It was washed with aq NaHCO₃ (1%, 2×300 mL), water (300 mL) and brine (300 mL), dried (Na₂SO₄), concentrated, and purified by reverse phase (C18) chromatography eluting with 0 to 95% ACN in water to give the product. LCMS (ES, m/z): 877.5 [M+H]⁺. ¹H-NMR: (400 MHz, DMSO-d₆): δ 12.01 (s, 1H), 8.92 (s, 1H), 8.11 (d, J=7.6 Hz, 2H), 7.66 (dt, J=42.3, 7.5 Hz, 3H), 7.32 (td J=7.2, 6.6, 2.9 Hz, 2H), 7.22-7.00 (m, 9H), 6.83-6.63 (m, 4H), 5.86 (ddt, J=52.8, 17.6, 6.9 Hz, 1H), 5.16 (td, J=17.7, 17.2, 8.8 Hz, 1H), 3.78-3.63 (m, 7H), 3.59-3.35 (m, 5H), 2.74 (t, J=5.9 Hz, 1H), 2.63 (t, J=5.9 Hz, 1H), 1.23-0.99 (m, 10H), 0.91 (d, J=6.7 Hz, 2H). ³¹P-NMR: (162 MHz, DMSO-d₆): δ 150.26, 149.60 (2 s, 1P).

Preparation 3. N-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: N-(9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a suspension of 2-amino-9-((2R,3 S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (Carbosynth catalog #ND10826, 1.50 g, 5.26 mmol) in pyridine (30 mL) at 0-5° C. was added TMSCl (2.86 g, 26.3 mmol), and the mixture was stirred at rt for 30 min. Then, isobutyric anhydride (2.50 g, 15.8 mmol) was added dropwise, and it was stirred for an additional for 2h. Then, MeOH (5.3 mL) was added. After 5 min, NH₄OH (10.5 mL) was added dropwise and stirring was continued for 30 min. The reaction mixture was concentrated under reduced pressure, and MeOH (2 mL) in CH₂Cl₂ (18 mL) was added to the residue. Insolubles were filtered off, and the filtrate was concentrated and purified by flash column chromatography with 2-10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 356.1 [M+H]⁺. ¹H-NMR: (400 MHz, DMSO-d₆): δ 12.11 (s, 1H), 11.68 (s, 1H), 8.28 (s, 1H), 5.98 (d, J=6.1 Hz, 1H), 5.85 (d, J=8.0 Hz, 1H), 5.24 (t, J=5.4 Hz, 1H), 5.14 (d, J=4.1 Hz, 0.5H), 5.01 (d, J=4.2 Hz, 0.5H), 4.87-4.69 (m, 1H), 4.26 (t, J=4.4 Hz, 0.5H), 4.19 (t, J=4.4 Hz, 0.5H), 3.61 (t, J=4.9 Hz, 2H), 2.77 (hept, J=6.8 Hz, 1H), 1.13 (d, J=6.7 Hz, 6H). ¹⁹F-NMR: (376 MHz, DMSO-d₆): δ −197.5 (s).

Step 2: N-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3 S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1.30 g, 3.66 mmol) was co-evaporated with pyridine (3×10 mL) and re-dissolved in pyridine (26 mL). To the solution at 0-5° C. was added DMTrCl (1.36 g, 4.02 mmol). It was stirred at rt for 3h and then, concentrated. CH₂Cl₂ (40 mL, with 1% Et₃N) was added, and it was washed with sat aq NaHCO₃ (15 mL), water (10 mL) and brine (10 mL). The organic solution was dried (Na₂SO₄), concentrated and purified by silica gel column chromatography using 0-10% MeOH in CH₂Cl₂ (1% Et₃N) to give the product. LCMS (ES, m/z): 656.2 [M−H]⁻. ¹H-NMR: (400 MHz, DMSO-d₆): δ 12.10 (s, 1H), 11.61 (s, 1H), 8.14 (s, 1H), 7.40-7.31 (m, 2H), 7.31-7.19 (m, 7H), 6.89-6.78 (m, 4H), 6.08 (d, J=6.1 Hz, 1H), 5.87 (d, J=7.3 Hz, 1H), 5.23 (dd, J=4.1, 1.8 Hz, 0H), 5.10 (d, J=4.4 Hz, 0H), 4.96 (dq, J=22.4, 5.9 Hz, 1H), 4.30 (dt, J=26.1, 4.6 Hz, 1H), 3.74 (d, J=1.1 Hz, 6H), 3.39 (dd, J=10.6, 5.7 Hz, 1H), 3.22 (dd, J=10.6, 3.8 Hz, 1H), 2.76 (p, J=6.8 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H). ¹⁹F-NMR: (376 MHz, DMSO-d₆): δ −198.1 (s, 1F).

The product of Preparation 3 may optionally be treated according to the procedures of Preparation 22, Steps 4 and 5 (below), to afford (2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate. LCMS (ES, m/z): 720 [M−H]⁻.

Preparation 4: N-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1: ((2R,3R,4S,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methyl benzoate

To a suspension of 8-azaguanine (5.14 g, 33.8 mmol) in anhydrous CH₃CN (100 mL) at rt was added dropwise (E)-trimethylsilyl N-(trimethylsilyl)acetimidate (16.53 mL, 67.6 mmol), then the mixture was stirred at 70° C. for 2h. The reaction was cooled to rt, and a solution of ((2R,3R,4S)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methyl benzoate (6.8 g, 16.90 mmol) in anhydrous CH₃CN (20 mL) was added followed by dropwise addition of tin(IV) chloride (67.6 mL, 67.6 mmol). The homogeneous solution was stirred at 70° C. for 2h. The reaction was cooled to rt and concentrated. The residue was dissolved in EtOAc (1000 mL) and neutralized by pouring into sat aq NaHCO₃ (500 mL). The organic layer was separated, and the aq layer was extracted with EtOAc (4×500 mL). The organic layers were combined and washed with water (3×700 mL), brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford title compound without further purification. LCMS (ES, m/z): 495.3 [M+H]⁺.

Step 2: ((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methyl benzoate

To a solution of ((2R,3R,4S,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methyl benzoate (8 g, 16.18 mmol) from Step 1 in anhydrous DMA (40 mL) at rt was added dropwise isobutyric anhydride (4.02 mL, 24.27 mmol). The mixture was stirred at 140° C. for 4h. The reaction was cooled and diluted with EtOAc (600 mL), washed with sat aq NH₄Cl (4×500 mL), brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by MPLC (220 g silica gel, eluting with a gradient of 100% hexanes to 100% ethyl acetate) to afford ((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methyl benzoate. LCMS (ES, m/z): 565.3 [M+H]⁺.

Step 3: N-(3-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of ((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methyl benzoate (6 g, 10.63 mmol) in THF (20 mL), CH₃OH (16 mL), and water (4 mL) at 0° C. was added 5N aqueous NaOH (4.89 mL, 24.45 mmol) and stirred for 1h. The reaction was neutralized with formic acid (1.223 mL, 31.9 mmol). The solvent was removed, and the residue was purified by MPLC (120 g, silica gel, eluting with a gradient of 100% CH₂Cl₂ to 20% CH₃OH/CH₂Cl₂) to afford N-(3-((2R,3 S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide. LCMS (ES, m/z): 357.2 [M+H]⁺.

Step 4: N-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of N-(3-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide (3 g, 8.42 mmol) in anhydrous pyridine (40 mL) at 0° C. was added 4,4′-dimethoxytrityl chloride (3.42 g, 10.10 mmol). The ice bath was removed, and the reaction mixture was allowed to reach RT and was stirred for 2h. The mixture was diluted with EtOAc (400 mL), washed with sat aq NaHCO₃ (100 mL), water (3×100 mL), brine, and dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by MPLC (120 g silica gel, eluting with a gradient of 100% CH₂Cl₂ to 15% CH₃OH/CH₂CH₂ to afford N-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide. LCMS (ES, m/z): 659.3 [M+H]⁺.

Preparation 5: N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

N-(3-((2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide was prepared according to procedures analogous to those described for Preparation 4 using the appropriately substituted ribose in Step 1. LCMS (ES, m/z): 659.4 [M+H]⁺.

Preparation 6: N-(3-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl) isobutyramide

N-(3-((2R,3R,5 S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide was prepared according to procedures analogous to those described for Preparation 4 using the appropriately substituted ribose in Step 1. LCMS (ES, m/z): 641.2 [M+H]⁺.

Preparation 7: 3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

Step 1: 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine

To a stirred mixture of freshly ground KOH (308 mg, 5.48 mmol) in acetonitrile (50 mL) was added tris(2-(2-methoxyethoxy)ethyl)amine (0.070 mL, 0.219 mmol). The reaction mixture was aged for 15 min at ambient temperature followed by addition of 7-nitro-3H-imidazo[4,5-b]pyridine (600 mg, 3.66 mmol) in a single portion. The resulting mixture was stirred at ambient temperature for 15 min. A solution of 2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-α-D-arabinofuranosyl bromide (1700 mg, 4.02 mmol) in acetonitrile (10 mL) was added dropwise and the resulting mixture was vigorously stirred at RT for 17h. The reaction mixture was diluted with sat aq ammonium chloride (80 mL) and extracted with DCM (3×150 mL). The organic extracts were combined, dried over sodium sulphate and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel; 120 g prepacked, (0-70% ethyl acetate/hexanes) to afford 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-3-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine. MS: 507 (M+H)⁺.

Step 2: 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-fl-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine (1380 mg, 2.72 mmol) in methanol (55 mL) at RT was added platinum(IV) oxide (61.9 mg, 0.272 mmol). The reaction mixture was placed under an atmosphere of hydrogen and stirred at RT for 72h. The catalyst was removed by filtration through a plug of CELITE. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography on silica gel; 80g prepacked, ((0-40% (3:1, ethyl acetate:ethanol)/hexanes) to afford 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine. MS: 477 (M+H)⁺.

Step 3: 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of 3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine (995 mg, 2.09 mmol) in methanol (36 mL) at ambient temperature was added ammonia (7N in methanol, 12 mL, 84.0 mmol). The resulting solution was brought to 80° C. and stirred for 5h. The reaction mixture was cooled to RT and concentrated under reduced pressure. The resulting residue was suspended in methanol/dichloromethane and sonicated until a solid precipitated out of solution. Solid was collected by filtering through a glass frit affording 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine. MS: 269 (M+H)⁺.

Step 4: 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine (550 mg, 2.05 mmol) in pyridine (6.5 mL) at RT was added TMSCl (2.62 mL, 20.5 mmol). The resulting solution was stirred for 1.5h followed by addition of benzyol chloride (0.357 mL, 3.08 mmol). After stirring for an additional hour, water (2.15 mL) was added to the reaction mixture, which was then stirred for 45 min. The reaction mixture was cooled to 0° C. and aqueous ammonia (28% w/w) (0.370 mL, 4.79 mmol) was added. The reaction mixture was returned to RT and stirred for 45 min and then concentrated under reduced pressure. The resulting residue was taken up in water (20 mL) and extracted with ethyl acetate (3×40 mL). The organic extracts were combined washed with brine, dried over sodium sulphate and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel; 120 g prepacked, (0-7% methanol/dichloromethane) to afford 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine. MS: 373 (M+H)⁺.

Step 5: 3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred mixture of 3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine (185 mg, 0.497 mmol) and 4 Å molecular sieves in pyridine (3 mL) at 0° C. was added 4,4′-dimethoxytrityl chloride (253 mg, 0.745 mmol) in a single portion. The reaction mixture was allowed to warm to RT and was stirred for 18h. Sieves were removed by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was taken up in a mixture of methanol/ether and added to water. The phases were separated, and the aqueous layer was extracted with ether (3 times). The organic extracts were combined, washed with brine, dried over sodium sulphate and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel; 40g prepacked, ((0-40% (3:1, ethyl acetate:ethanol)/hexanes) to afford 3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine. MS: 675 (M+H)⁺.

Preparation 8: 1-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

Step 1: ((2R,3S,4S)-4-acetoxy-3-fluoro-5-(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-2-yl)methyl benzoate

To a suspension of 4-(benzyloxy)-1H-imidazo[4,5-c]pyridine (0.795 g, 3.53 mmol) and (3S,4S,5R)-5-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (1 g, 2.94 mmol) in ACN (20 mL) and CH₂Cl₂ (10 mL) at 0° C. under Ar was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (1.34 g, 8.815 mmol). The resulting mixture was stirred at 0° C. for 30 min. Then, trimethylsilyl trifluoromethanesulfonate (3.92 g, 17.65 mmol) was added to the solution, and it was stirred at 0° C. for 30 min. Then, it was heated to 80° C. for 16h. The reaction was cooled to rt and sat aq NaHCO₃ (10 mL) and water (30 mL) were added. It was extracted with EtOAc (3×50 mL). The combined organic layers was washed with brine, dried over (Na₂SO₄), concentrated, and the residue was purified by silica gel column chromatography, eluted with 1 to 10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 416.3 [M+H]⁺.

Step 2: 1-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

To a solution of ((2R,3S,4S)-4-acetoxy-3-fluoro-5-(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-2-yl)methyl benzoate (2.5 g, 5.3 mmol) in MeOH (10 mL) was added sodium methanolate (3.47 g, 21.2 mmol). The solution was stirred at rt for 1h. It was neutralized with AcOH, and the solution was concentrated. The residue was purified by reverse phase (AQ C18) chromatography eluted with 0 to 30% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 270.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 11.31 (s, 1H), 8.06 (s, 1H), 7.23 (d, J=7.1 Hz, 1H), 6.62 (d, J=7.1 Hz, 1H), 6.37 (d, J=2.9 Hz, 1H), 5.85 (d, J=2.8 Hz, 1H), 5.22-4.98 (m, 2H), 4.54 (d, J=17.7 Hz, 1H), 4.28 (dtd, J=29.6, 6.0, 3.1 Hz, 1H), 3.93-3.62 (m, 2H).

Step 3: 1-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

To a stirred solution of 1-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-1H-imidazo[4,5-c]pyridin-4(5H)-one (340 mg, 1.26 mmol) in pyridine (3 mL) at rt was added 4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (426 mg, 1.1 mmol).

It was stirred for 4h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluted with 1 to 10% MeOH in CH₂Cl₂ (0.5% Et₃N) to give the product. LCMS (ES, m/z): 572.3 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 11.32 (d, J=5.9 Hz, 1H), 7.95 (s, 1H), 7.41 (d, J=7.8 Hz, 2H), 7.37-7.15 (m, 8H), 6.86 (dd, J=10.5, 8.6 Hz, 4H), 6.60 (d, J=7.1 Hz, 1H), 6.50-6.36 (m, 1H), 5.92 (d, J=2.6 Hz, 1H), 5.77 (s, 1H), 5.27-5.06 (m, 1H), 4.65-4.42 (m, 2H), 3.73 (d, J=2.6 Hz, 6H), 3.30-3.24 (m, 1H).

Preparation 9: N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

Step 1: (3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol

To a stirring mixture of 7-bromoimidazo[2,1-f][1,2,4]triazin-4-amine (41 g, 0.19 mol) in THF (0.50 L) at 0° C. was added MeMgBr (3.0M in THF, 66 mL, 0.19 mol) dropwise to maintain the internal temperature below 10° C. Bis(chlorodimethylsilyl)ethane (41 g, 190 mmol) was added in one portion. MeMgBr (3.0M in diethyl ether, 66 mL, 0.19 mol) was then added dropwise to maintain the internal temperature below 10° C. i-PrMgCl—LiCl (1.3 M in THF, 0.16 L, 0.21 mol) was added while maintaining the internal temperature below 10° C. A mixture of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one (160 g, 0.38 mol) in THF was added dropwise at 0° C., and the mixture was then allowed to warm to rt and was stirred for 12h. The mixture was diluted with saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (3×1000 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by column chromatography (column height: 2500 mm, diameter: 1000 mm, 25% to 75% ethyl acetate gradient in hexanes) to afford (3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-tetrahydrofuran-2-ol.

Step 2: 7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine

To a stirring mixture of (3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (64 g, 0.12 mmol) in DCM (1.3 L) at 0° C. was added triethylsilane (81 g, 0.69 mol), and then boron trifluoride diethyl etherate (21 g, 0.15 mol). The mixture was then allowed to warm to 25° C., and the mixture was stirred for 1h. More boron trifluoride diethyl etherate (57 g, 0.40 mol) was added, and the mixture was then heated to 35° C. for 4h. Upon cooling to rt, the mixture was quenched with saturated aqueous sodium bicarbonate (200 mL) and then extracted with ethyl acetate (3×300 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by column chromatography (15-75% ethyl acetate gradient in hexanes) to afford 7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine. MS (ES, m/z)=538 [M+H]+.

Step 3: (3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol

To a stirring mixture of 7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine (12 g, 22 mmol) in DCM (850 mL) at −78° C. was added boron trichloride (18 g, 0.16 mol) dropwise. Upon completion, the mixture was stirred at −78° C. for 3h. After 3h, the mixture was quenched with methanol (50 mL) at −78° C., and the mixture was allowed to warm to 25° C. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (9-25% methanol gradient in dichloromethane) to afford (3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol.

Step 4. (6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol

To a stirred mixture of (2S,3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (4.0 g, 15 mmol) in pyridine (0.10 L) was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (5.8 mL, 18 mmol). After 3h, the mixture was diluted with toluene (50 mL) and then concentrated. The resulting mixture was taken up in DCM and methanol and then silica gel (40g) was added. The mixture was concentrated, placed under vacuum for 1h and then purified by column chromatography (0-80% ethyl acetate gradient in hexanes) to afford (6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol. MS (ES, m/z)=510 [M+H]+.

Step 5: O-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl) 1H-imidazole-1-carbothioate

To a mixture of (6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol (6.45 g, 12.7 mmol) in acetonitrile (63.0 mL) and pyridine (63.0 mL) was added 1,1′-thiocarbonyldiimidazole (2.71 g, 15.2 mmol). After 90 min, more 1,1′-thiocarbonyldiimidazole (2.71 g, 15.2 mmol) was added, and the mixture was stirred overnight. After stirring overnight, the mixture was concentrated and purified by column chromatography (0-100% ethyl acetate gradient in hexanes) to afford O-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl) 1H-imidazole-1-carbothioate. MS (ES, m/z)=620 [M+H]+.

Step 6: 7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine

To a mixture of O-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl) (5.65 g, 9.11 mmol) in toluene (91.0 mL) was added 2,2′-azobis(2-methylpropionitrile (0.300 g, 1.82 mmol) and tris(trimethylsilyl)silane (4.22 mL, 13.7 mmol). The mixture was heated to 85° C. for 30 min. After 30 min, the mixture was allowed to cool to rt and placed directly on the column and purified (0-80% ethyl acetate gradient in hexanes) to afford 7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine. MS (ES, m/z)=494 [M+H]+ 494.

Step 7: N-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture of 7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine (15.7 g, 31.8 mmol) in pyridine (64.0 mL) was added benzoyl chloride (11.0 mL, 95.0 mmol), and the mixture was heated to 50° C. for 45 min. After 45 min, the mixture was allowed to cool to rt. After cooling, a precipitate formed and was filtered off. The filtrate was diluted with DCM (50 mL) and toluene (50 mL). The mixture was concentrated to about 50 mL. The mixture was filtered, and the solids washed with DCM. The filtrate and washes were combined, loaded onto a column, and purified (0-50% ethyl acetate gradient in hexanes) to afford N-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide. MS (ES, m/z)=702 [M+H]+.

Step 8: N-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture of N-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide (10 g, 14 mmol) in tetrahydrofuran (0.14 L) was added TBAF ((1.0M in THF, 29 mL, 29 mmol), and the mixture was stirred for 1h. After 1h, the mixture was concentrated and purified by column chromatography to afford N-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro-furan-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide. MS (ES, m/z)=356 [M+H]+.

Step 9: N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture of N-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide (6.1 g, 17 mmol) in pyridine (86 mL) at 0° C. was added 4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (5.8 g, 17 mmol), and the mixture was allowed to warm to RT overnight. After stirring overnight, the mixture was diluted with toluene and then concentrated under reduced pressure to afford the crude product. The crude product was purified by silica gel chromatography (0-100% ethyl acetate gradient in hexanes) to afford N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide. MS (ES, m/z)=658 [M+H]+.

Preparation 10: (2R,3R,4S,5R)-2-(4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

Step 1: (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate

To a suspension of 4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridine (1.0 g, 6.5 mmol) and (3R,4R,5R)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triyl triacetate (3.1 g, 9.7 mmol) in nitromethane (50 mL) was added BF₃.Et₂O (1.23 mL, 9.7 mmol), and the resulting mixture was heated at 50° C. for 2h. The reaction mixture was cooled to rt, diluted with 100 mL of DCM and washed with sat aq. NaHCO₃ (100 mL) and brine (100 mL). The separated organic layer was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-100% EtOAc:EtOH (3:1)/Hexane. LCMS (ES, m/z): 413.07 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.40 (d, J=5.8 Hz, 1H), 7.56 (d, J=5.8 Hz, 1H), 6.43 (d, J=4.1 Hz, 1H), 6.17 (dd, J=5.3, 4.1 Hz, 1H), 5.74 (t, J=5.3 Hz, 1H), 5.32 (s, 1H), 4.58 (ddd, J=5.3, 3.9, 2.9 Hz, 1H), 4.42 (dd, J=12.5, 3.0 Hz, 1H), 4.25 (dd, J=12.5, 3.9 Hz, 1H), 2.17 (d, J=18.8 Hz, 6H), 2.03 (s, 3H).

Step 2: (2R,3R,4S,5R)-2-(4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

To a solution of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate (1.2 g, 2.9 mmol) in MeOH (8.3 mL) was added a 7N solution of ammonia in MeOH (8.3 mmol, 58.1 mmol). The reaction mixture was stirred at 150° C. in a sealed Q-Tube™ Pressure Tube Reactor for 18h. Excess solvent was removed under reduced pressure, and the residue was purified by a reverse phase silica gel column, eluting with 0-10% Acetonitrile/H₂O containing 0.05% TFA. LCMS (ES, m/z): 268.17 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 13.87 (s, 2H), 9.34 (s, 4H), 7.89 (d, J=7.1 Hz, 2H), 7.51 (d, J=7.0 Hz, 2H), 7.42 (s, 4H), 7.32 (s, 6H), 7.22 (s, 5H), 6.69 (s, 2H), 6.30 (d, J=4.9 Hz, 2H), 5.72 (s, 2H), 5.41 (s, 2H), 4.75-4.67 (m, 0H), 4.65 (t, J=5.0 Hz, 2H), 4.27 (t, J=4.6 Hz, 2H), 4.07 (q, J=4.2 Hz, 2H), 3.86-3.67 (m, 2H), 3.69-3.58 (m, 2H), 3.54 (dd, J=12.0, 4.7 Hz, 2H), 3.48-3.38 (m, 1H), 3.28 (s, 1H), 3.23 (s, 0H), 1.76 (d, J=0.5 Hz, 5H), 1.11 (dt, J=26.0, 7.1 Hz, 1H).

Preparation 11: 9-{3-azido-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-β-D-ribofuranosyl}-2-[(2-methylpropanoyl)amino]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures (Nucleosides, Nucleotides & Nucleic Acids 2005, 24(10-12), 1707-1727).

Preparation 12: 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-3-fluoro-β-D-xylofuranosyl}-2-[(2-methylpropanoyl)amino]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures (Tetrahedron Letters, 1989, 30(24), 3171-3174).

Preparation 13: 1-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-3-fluoro-β-D-ribofuranosyl}-6-[(2-methylpropanoyl)amino]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures (WO2002057425).

Preparation 14: N-(3-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

Step 1: (((2R,3R)-3-(benzoyloxy)-5-((diphenoxyphosphoryl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate

To ((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate (20.0 g, 52.9 mmol) in toluene (150 mL) at 0° C. were added Et₃N (7.74 mL, 55.5 mmol) and diphenyl phosphoryl chloride (12.1 mL, 58.2 mmol) in toluene (20 mL) dropwise. The reaction was warmed to rt and stirred for 3h. LCMS showed completion of the reaction (m/e=611). Water (30 mL) and aq HCl (1 M, 5 mL) were added. Layers were separated, and the aq layer was extracted with CH₂Cl₂ (2×30 mL). The combined organic solution was washed with sat aq NaHCO₃ (20 mL), and Brine (20 mL), dried (MgSO₄), and concentrated. It was purified by silica column chromatography eluting with 0 to 30% EtOAc in Hex to give the product. LCMS (ES, m/z): 611.3 [M+H]⁺.

Step 2: ((2R,3R,5R)-3-(benzoyloxy)-5-bromo-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate

To ((2R,3R)-3-(benzoyloxy)-5-((diphenoxyphosphoryl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate (23.8 g, 39.0 mmol) at 0° C. was added HBr in acetic acid (33%, 51.2 mL, 292 mmol). It was stirred as it warmed to rt. After 6h, LCMS showed completion of the reaction (m/e=441 and 443). CH₂Cl₂ (200 mL) was added, and the organic layer was washed with water (2×50 mL), sat aq NaHCO₃ (2×50 mL) and brine (50 mL). It was dried (MgSO₄) and concentrated to give the crude product. It was used in the next step without purification. LCMS (ES, m/z): 441.1, 443.1 [M+H]⁺.

Step 3: (2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-3-yl benzoate

To a mixture of 3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (3.21 g, 23.6 mmol) in NMP (60 mL) was added NaH (60%, 0.979 g, 24.5 mmol). The mixture was vigorously stirred, and after 1h, generation of bubbles had completely ceased. To the mixture was added to ((2R,3R,5R)-3-(benzoyloxy)-5-bromo-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate (neat, 8.00 g, 18.1 mmol). The mixture was stirred vigorously for 30 min. Then, it was heated to 90° C. for 5h. It was cooled to rt, and CH₂Cl₂ (300 mL) and water (150 mL) were added. The phases were separated, and the organic layer was washed with water (8×150 mL) and brine (50 mL), dried (MgSO₄) and concentrated. The residue was purified by silica column chromatography eluting with 0% to 30% EtOAc to give the desired product. LCMS (ES, m/z): 497.1 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.44 (s, 1H), 8.15-8.08 (m, 2H), 8.08-7.99 (m, 2H), 7.63 (ddt, J=8.7, 7.1, 1.3 Hz, 1H), 7.56-7.44 (m, 3H), 7.40-7.32 (m, 2H), 6.79-6.68 (m, 2H), 6.04 (bs, 2H), 4.92-4.84 (m, 1H), 4.80-4.72 (m, 2H).

Step 4. (2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To (2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy) methyl)-4,4-difluorotetrahydrofuran-3-yl benzoate (1.05 g, 2.11 mmol) was added ammonia in MeOH (7N, 9.0 mL, 63 mmol), and the mixture was stirred for 24h. LCMS showed completion of reaction (m/e=289). It was concentrated and purified by silica column chromatography eluting with 0 to 15% MeOH in CH₂Cl₂ to give the desired product. LCMS (ES, m/z): 289.1 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 8.65 (s, 1H), 8.36 (s, 1H), 8.32 (s, 1H), 6.65 (d, J=12.4 Hz, 1H), 6.40 (d, J=6.7 Hz, 1H), 5.03 (dd, J=6.4, 5.3 Hz, 1H), 4.83 (dq, J=16.5, 8.9 Hz, 1H), 4.00 (t, J=6.7 Hz, 1H), 3.77 (ddd, J=12.3, 5.2, 2.5 Hz, 1H), 3.68 (dt, J=12.5, 6.3 Hz, 1H).

Step 5. N-(3-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of (2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (0.57 g, 2.0 mmol) in pyridine (15 mL) at 0° C. was added TMSCl (0.55 mL, 4.3 mmol). It was warmed to rt and stirred for 1h. Then, it was recooled to 0° C. and BzCl (0.34 mL, 2.9 mmol) was added dropwise. The reaction was slowly warmed rt over 2h. LCMS showed completion of reaction (m/e=393, 497). It was cooled to 0° C., and ammonium hydroxide (28%, 1.1 mL, 7.9 mmol) was added. After 30 min, it was concentrated and purified by silica column chromatography eluting with 0 to 10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 393.3 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 12.02 (s, 1H), 9.01 (s, 1H), 8.13-8.07 (m, 2H), 7.73-7.66 (m, 1H), 7.64-7.55 (m, 2H), 6.86 (d, J=11.6 Hz, 1H), 6.46 (d, J=6.7 Hz, 1H), 5.01 (dd, J=6.3, 5.4 Hz, 1H), 4.86 (dt, J=16.7, 8.3 Hz, 1H), 4.04 (ddd, J=8.8, 6.2, 2.6 Hz, 1H), 3.78 (ddd, J=12.3, 5.4, 2.7 Hz, 1H), 3.68 (dt, J=12.4, 6.2 Hz, 1H).

Step 6. N-(3-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of N-(3-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide (0.68 g, 1.7 mmol) in pyridine (15 mL) at 0° C. was added DMTrCl (0.65 g, 1.9 mmol). It was stirred at rt for 1h. LCMS showed a peak with the desired mass (m/e=695). It was partly concentrated (to 5 mL), and EtOAc (20 mL) and water (10 mL) were added. The phases were separated, and the aqueous layer was extracted with EtOAc (2×10 mL). The combined organic portions were washed with brine, dried (MgSO₄), concentrated and purified by silica column chromatography eluting with 0% to 60% EtOAc in Hex to give the product. LCMS (ES, m/z): 695.2 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 12.05 (s, 1H), 8.93 (s, 1H), 8.11 (d, J=7.6 Hz, 2H), 7.70 (m, 1H), 7.60 (t, J=7.7 Hz, 2H), 7.38-7.32 (m, 2H), 7.25-7.13 (m, 7H), 6.96 (d, J=11.9 Hz, 1H), 6.84-6.73 (m, 4H), 6.48 (d, J=6.9 Hz, 1H), 4.97 (dq, J=16.7, 8.3 Hz, 1H), 4.23 (m, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.42-3.36 (m, 1H), 3.32-3.28 (m, 1H).

Preparation 15: N-(7-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

Step 1. ((2R,3R,5R)-3-(benzoyloxy)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate

To a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (4.06 g, 26.4 mmol) and ((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate (10.0 g, 26.4 mmol) and triphenylphosphine (20.80 g, 79 mmol) in THF (100 mL) was added (E)-diazene-1,2-diylbis(piperidin-1-ylmethanone) (20.01 g, 79 mmol) dropwise. It was slowly warmed to rt. After stirring for 2h, the mixture was concentrated. The residue was purified by silica gel column chromatography using 0-20% EtOAc in Petroleum Ether to give the product. LCMS (ES, m/z): 514.3 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.71 (s, 1H), 8.08-7.87 (m, 5H), 7.76-7.42 (m, 6H), 6.98 (dd, J=10.3, 7.9 Hz, 1H), 6.84 (d, J=3.8 Hz, 1H), 6.29 (ddd, J=10.0, 6.0, 3.8 Hz, 1H), 5.44 (q, J=5.5 Hz, 1H), 4.79-4.60 (m, 2H).

Step 2. (2R,3R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

A solution of ((2R,3R,5R)-3-(benzoyloxy)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl benzoate (1.9 g, 3.70 mmol) in NH₃/2-propanol (saturated at −78° C., 100 mL) was stirred at 90° C. for 16h. It was cooled to rt, concentrated, and purified by silica gel column chromatography using 0-10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 287.1 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.06 (s, 1H), 7.40-7.25 (m, 1H), 7.14 (s, 2H), 6.82 (d, J=5.9 Hz, 1H), 6.60 (d, J=3.7 Hz, 1H), 6.34 (dd, J=16.2, 2.1 Hz, 1H), 4.88 (t, J=5.7 Hz, 1H), 4.27 (ddd, J=9.9, 6.2, 3.6 Hz, 1H), 4.08 (dq, J=6.8, 4.1, 3.5 Hz, 1H), 3.78 (dt, J=11.1, 5.4 Hz, 1H), 3.62 (dt, J=11.4, 5.8 Hz, 1H).

Step 3: N-(7-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution of (2R,3R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (900 mg, 3.14 mmol) in pyridine (15 mL) at 0° C. was added chlorotrimethylsilane (3.42 g, 31.4 mmol). It was warmed to rt over 1h and benzoyl chloride (663 mg, 4.72 mmol) was added dropwise. After 2h, NH₄OH (28%, 15.00 mL) was added, and it was stirred for 0.5h. The resulting mixture was concentrated, and the residue was purified by silica gel column chromatography using 0-10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 391.1 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.61 (s, 1H), 8.08-7.98 (m, 2H), 7.97-7.91 (m, 1H), 7.67-7.51 (m, 2H), 7.50-7.47 (m, 1H), 6.70 (d, J=3.9 Hz, 1H), 6.56 (dd, J=15.6, 1.5 Hz, 1H), 4.32 (dd, J=9.4, 3.6 Hz, 1H), 4.16 (d, J=5.6 Hz, 1H), 3.80 (dd, J=11.5, 5.2 Hz, 1H), 3.66 (dd, J=11.5, 6.5 Hz, 1H), 3.12 (s, 2H).

Step 4. N-(7-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution of N-(7-((2S,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (1.1 g, 2.82 mmol) in pyridine (12 mL) at 0° C. was added 4,4′-(chloro(phenyl)methylene)-bis(methoxybenzene) (0.955 g, 2.82 mmol). It was warmed to RT and stirred for 3h. The mixture was concentrated. The product was purified by silica gel column chromatography using 0-10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 693.3 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 11.25 (s, 1H), 8.67 (s, 1H), 8.09-8.01 (m, 2H), 7.60-7.48 (m, 2H), 7.47-7.36 (m, 3H), 7.33-7.18 (m, 7H), 6.91-6.78 (m, 4H), 6.75-6.57 (m, 3H), 5.76 (s, 1H), 4.55-4.33 (m, 2H), 3.74 (s, 6H), 3.44 (t, J=8.7 Hz, 1H), 3.35 (s, 1H).

Preparation 16: N-(9-((2R,3S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: (2R,4S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluoro-2-hydroxybutyl benzoate

To a solution of (2R,4S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluorobutane-1,2-diol (3.50 g, 10.5 mmol) in CH₂Cl₂ (52 mL) and pyridine (26 mL) at −70° C. was added benzoyl chloride (1.48 g, 10.5 mmol) in CH₂Cl₂ (11 mL) dropwise over 50 min. After 2h, methanol (150 mL) was added. The mixture was stirred at RT for 0.5h. Water (200 mL) was added. Layers were separated, and the aq layer was extracted with ether (4×150 mL). The combined organic layers were washed with aq HCl (1 N, 2×150 mL), sat aq NaHCO₃ (2×150 mL) and brine (2×150 mL). It was dried (Na₂SO₄), concentrated and purified by silica gel column chromatography (EtOAc/petroleum ether=1/10) to give the product. ¹H-NMR (400 MHz, CDCl₃): δ 8.11-8.01 (m, 2H), 7.61-7.55 (m, 1H), 7.48-7.31 (m, 7H), 4.89-4.53 (m, 4H), 4.46-4.41 (m, 1H), 4.40-4.29 (m, 1H), 4.17-4.02 (m, 2H), 3.98-3.84 (m, 0.5H), 3.74-3.66 (m, 0.5H), 1.46 (s, 3H), 1.28 (s, 3H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −106.8 (d, J=270.7 Hz, 1F), −119.2 (d, J=270.7 Hz, 1F).

Step 2: ((2R,4S)-4-(benzyloxy)-3,3-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate

(2R,4 S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluoro-2-hydroxybutyl benzoate (3.00 g, 6.87 mmol) was dissolved in aq AcOH (75%, 66 mL). It was stirred at 50° C. for 3h. It was partly concentrated. The residue was redissolved in acetone (33 mL). To the solution at rt was added sodium periodate (1.20 g, 5.61 mmol) in water (33 mL). After 1.5h, the solid formed was filtered off and washed with acetone. The filtrate was concentrated. Water and CH₂Cl₂ were added, and layers were separated. The aq layer was extracted with CH₂Cl₂ (4×150 mL). The combined organic layers were dried (Na₂SO₄), concentrated, and purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/5) to give the product. ¹H-NMR (400 MHz, CDCl₃): δ 8.05-8.01 (m, 2H), 7.59-7.52 (m, 1H), 7.46-7.35 (m, 7H), 5.49-5.42 (m, 1H), 4.99-4.72 (m, 1H), 4.67-4.47 (m, 4H), 4.11-3.80 (m, 1H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −117.1 (d, J=240.6 Hz, 1F), −117.9 (d, J=251.9 Hz, 1F).

Step 3: ((2R,4S)-5-acetoxy-4-(benzyloxy)-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate

To a solution of ((2R,4S)-4-(benzyloxy)-3,3-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate (2.40 g, 6.59 mmol) in CH₂Cl₂ (66 mL) at rt was added N,N-dimethylpyridin-4-amine (0.080 g, 0.659 mmol) and acetic anhydride (4.03 g, 39.5 mmol) dropwise. After 6h, it was quenched by addition of sat aq NaHCO₃ (30 mL). Layers were separated, and the aq layer was extracted with CH₂Cl₂ (3×150 mL). The combined organic layers were washed with water (2×150 mL) and brine (2×100 mL), dried (Na₂SO₄), concentrated and purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/7) to give the product. ¹H-NMR (300 MHz, CDCl₃): δ 8.08-7.89 (m, 2H), 7.61-7.48 (m, 1H), 7.47-7.25 (m, 7H), 6.15 (d, J=6.0 Hz, 1H), 4.89-4.75 (m, 1H), 4.73-4.40 (m, 4H), 4.18-4.02 (m, 1H), 1.98 (s, 3H). ¹⁹F-NMR: (282 MHz, CDCl₃): δ −116.5 (d, J=248.2 Hz, 1F), −120.9 (d, J=248.2 Hz, 1F).

Step 4: ((2R,4S)-5-acetoxy-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)methyl benzoate

To a solution of ((2R,4S)-5-acetoxy-4-(benzyloxy)-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate (2.50 g, 6.15 mmol) in EtOAc (60 mL) was added sodium bromate (5.57 g, 36.9 mmol) in water (46 mL). The mixture was stirred vigorously, and to it was added sodium dithionite (6.43 g, 36.9 mmol) in water (92 mL) dropwise over 1h. After 5h, layers were separated, and the aq layer was extracted with EtOAc (5×150 mL). The combined organic layers were washed with sat aq Na₂S₂O₃ (2×150 mL) and brine (2×150 mL), dried (Na₂SO₄), concentrated, and purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/5) to give the product. ¹H-NMR (300 MHz, CDCl₃): δ 8.10-7.89 (m, 2H), 7.63-7.50 (m, 1H), 7.48-7.30 (m, 2H), 6.09 (d, J=6.0 Hz, 1H), 4.71-4.42 (m, 3H), 4.36-4.26 (m, 1H), 2.04 (s, 3H). 19F-NMR: (282 MHz, CDCl₃): δ −119.5 (d, J=248.2 Hz, 1F), −122.0 (d, J=248.2 Hz, 1F).

Step 5: (3S,5R)-5-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-2,3-diyl diacetate

To a solution of ((2R,4S)-5-acetoxy-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)methyl benzoate (2.00 g, 6.32 mmol) in CH₂Cl₂ (84 mL) at rt was added DMAP (0.08 g, 0.632 mmol) and acetic anhydride (3.87 g, 37.9 mmol) dropwise. After 6h, it was quenched by addition of sat aq NaHCO₃ (30 mL). Layers were separated, and the aq layer was extracted with CH₂C₁₂ (3×140 mL). The combined organic layers were washed with water (2×140 mL) and brine (2×140 mL), dried (Na₂SO₄), concentrated and purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/6) to give the product. ¹H-NMR (400 MHz, CDCl₃): δ 8.11-8.01 (m, 2H), 7.63-7.54 (m, 1H), 7.50-7.41 (m, 2H), 6.20 (d, J=4.0 Hz, 1H), 5.39 (d, J=8.0 Hz, 1H), 4.69-4.48 (m, 3H), 2.23 (s, 3H), 2.08 (s, 3H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −117.6 (d, J=251.9 Hz, 1F), −119.5 (d, J=251.9 Hz, 1F).

Step 6: ((2R,4S,5R)-4-acetoxy-5-(6-chloro-2-isobutyramido-9H-purin-9-yl)-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate

To a solution of (3 S,5R)-5-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-2,3-diyl diacetate (2.20 g, 6.14 mmol) and N-(6-chloro-9H-purin-2-yl)isobutyramide (1.77 g, 7.37 mmol) in ACN (80 mL) at 0° C. was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (2.80 mL, 18.4 mmol). After 0.5h, trimethylsilyl trifluoromethanesulfonate (6.82 mL, 36.8 mmol) was added dropwise to the reaction at 0° C. After 0.5h, it was heated at 80° C. for 16h. The reaction was then quenched by the addition of water (150 mL). Layers were separated, and the aq layer was extracted with EtOAc (3×150 mL). The combined organic layers were washed with sat aq NaHCO₃ (2×150 mL) and brine (2×150 mL), dried (Na₂SO₄), concentrated and purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/1) to give the product. ¹H-NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 8.08-7.98 (m, 2H), 7.64-7.53 (m, 1H), 7.51-7.40 (m, 2H), 6.25 (d, J=4.0 Hz, 1H), 5.98-5.93 (m, 1H), 4.85-4.53 (m, 3H), 2.92-2.80 (m, 1H), 2.22 (s, 3H) 1.28 (d, J=4.0 Hz, 6H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −116.7 (d, J=248.2 Hz, 1F), −118.1 (d, J=248.2 Hz, 1F).

Step 7: ((2R,4S,5R)-5-(2-acetamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-acetoxy-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate

To a solution of ((2R,4S,5R)-4-acetoxy-5-(6-chloro-2-isobutyramido-9H-purin-9-yl)-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate (1.80 g, 3.35 mmol) in AcOH (29 mL) was added Sodium acetate (1.37 g, 16.7 mmol) and acetic anhydride (29 mL). The reaction was stirred at 125° C. for 2.5h. It was cooled to rt, and MeOH (50 mL) was added. The mixture was concentrated under reduced pressure, and the residue was coevaporated with ethanol (2×50 mL). DCM (150 mL) and water (150 mL) were added, and layers were separated. The organic phase was washed with sat aq NaHCO₃ (2×150 mL), dried (Na₂SO₄), and concentrated to give the product. LCMS (ES, m/z): 492.1 [M+H]⁺.

Step 8: 2-amino-9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

To ((2R,4S,5R)-5-(2-acetamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-acetoxy-3,3-difluorotetrahydrofuran-2-yl)methyl benzoate (neat, 1.80 g, 3.66 mmol) was added NH₃ in MeOH (7 M, 90 mL, Me0H). It was stirred at rt for 60h. It was concentrated and purified by reverse phase (C18) chromatography eluting with 5-20% MeCN in aq NH₄HCO₃ (5 mM) to give the product. ¹H-NMR (300 MHz, DMSO-d₆): δ 10.7 (s, 1H), 7.95 (s, 1H), 6.56-6.44 (m, 3H), 5.62 (d, J=6.0 Hz, 1H), 5.32 (t, J=5.4 Hz, 1H), 4.90-4.77 (m, 1H), 4.23-4.08 (m, 1H), 3.68-3.52 (m, 2H). ¹⁹F-NMR: (282 MHz, DMSO-d₆): δ −113.1 (d, J=234.1 Hz, 1F), −121.8 (d, J=234.1 Hz, 1F).

Step 9. N-(9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3 S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (800 mg, 2.64 mmol) was co-evaporated with pyridine (3×3 mL). It was re-suspended in pyridine (13 mL). To the mixture at 0° C. was added chlorotrimethylsilane (1.686 mL, 13.19 mmol) dropwise. It was warmed to rt and stirred for 2h. The reaction was cooled to 0° C., and isobutyric anhydride (0.656 mL, 3.96 mmol) was added dropwise. It was warmed to rt, stirred for 2h, and then water (4 mL) and NH₄OH (8 mL) were added to the reaction. After 30 min, it was concentrated. The residue was purified by flash column chromatography with 0-10% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 374.1 [M+H]⁺. ¹H-NMR: (300 MHz, DMSO-d₆) δ 12.11 (s, 1H), 11.69 (s, 1H), 8.30 (s, 1H), 6.58 (d, J=6.0 Hz, 1H), 5.74 (dd, J=9.0, 3.0 Hz, 1H), 5.33 (t, J=6.0 Hz, 1H), 4.96-4.83 (m, 1H), 4.26-4.17 (m, 1H), 3.72-3.62 (m, 2H), 2.80-2.71 (m, 1H), 1.11 (d, J=9.0 Hz, 6H).

Step 10: N-(9-((2R,3S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3 S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (640 mg, 1.714 mmol) was co-evaporated with pyridine (3×3 mL) and then re-suspended in pyridine (5.7 mL). To the suspension was added 4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (639 mg, 1.886 mmol), and it was stirred for 16h. Then, it was concentrated and then, co-evaporated with toluene (3×20 mL). The crude was purified by silica column chromatography eluting with 1 to 30% MeOH in CH₂Cl₂ (containing 1% Et₃N) to give the product. LCMS (ES, m/z): 676.3 [M+H]⁺. ¹H-NMR: (300 MHz, DMSO-d₆) δ 12.11 (s, 1H), 11.61 (s, 1H), 8.15 (s, 1H), 7.34 (J=6.0, 3.0 Hz, 2H), 7.28-7.19 (m, 7H), 6.85-6.80 (m, 4H), 6.71 (d, J=6.0 Hz, 1H), 5.78 (d, J=6.0 Hz, 1H), 5.13-5.05 (m, 1H), 4.46-4.39 (m, 1H), 3.71 (s, 6H), 3.46-3.40 (m, 1H), 3.22-3.18 (m, 1H), 2.78-2.70 (m, 1H), 1.11 (d, J=9.0 Hz, 6H).

Preparation 17: N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: 2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-hydroxytetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1,9-dihydro-6H-purin-6-one

To a stirred suspension of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1H-purin-6(9H)-one (15 g, 50.1 mmol) in DMF (150 mL) at 0° C. under Ar was injected di-tert-butylsilanediyl bis(trifluoromethanesulfonate) (26.5 g, 60.1 mmol). The resulting solution was stirred at rt for 1h. It was used for the next reaction step directly without purification. LCMS (ES, m/z): 440.2 [M+H]⁺.

Step 2: 2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy) tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1,9-dihydro-6H-purin-6-one

To the reaction mixture from the previous step at 0° C. was added 1H-imidazole (17.05 g, 251 mmol) in one portion. The mixture was stirred rt for 0.5h. tert-butylchlorodimethylsilane (15.10 g, 100 mmol) was added to the mixture, and it was stirred at 60° C. for 16h. Then, the volatile components were removed under reduced pressure. The solid was suspended in cold methanol (75 mL), filtered, and washed with cold methanol (2×30 mL). The solid was kept under reduced pressure to give the product. LCMS (ES, m/z): 554.4 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 10.80 (s, 1H), 7.98 (s, 1H), 6.49 (s, 2H), 5.53 (s, 1H), 4.46 (d, J=3.2 Hz, 1H), 4.42 (d, J=9.9 Hz, 1H), 4.34 (dd, J=9.9, 4.7 Hz, 1H), 4.21 (t, J=10.5 Hz, 1H), 3.70-3.64 (m, 1H), 1.04 (s, 9H), 1.00 (s, 9H), 0.92 (s, 9H), 0.19 (s, 3H), 0.11 (s, 3H).

Step 3: N-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1H-purin-6(9H)-one (29.1 g, 52.5 mmol) was co-evaporated with dry pyridine (3×50 mL) and re-dissolved in pyridine (70 mL) and dichloromethane (210 mL). The mixture was charged with Ar and cooled to 0° C. To the mixture was added isobutyryl chloride (11.20 g, 105 mmol). It was stirred at rt for 4h. It was concentrated under reduced pressure. The solid was suspended in cold methanol (100 mL), filtered, and washed with cold methanol (3×50 mL). The solid was kept under reduced pressure to give the product. LCMS (ES, m/z): 624.1 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.13 (s, 1H), 11.39 (s, 1H), 8.32 (s, 1H), 5.61 (s, 1H), 4.66 (d, J=3.4 Hz, 1H), 4.48 (d, J=9.9 Hz, 1H), 4.38-4.33 (m, 1H), 4.21 (t, J=9.9 Hz, 1H), 3.76-3.70 (m, 1H), 2.84-2.80 (m, 1H), 1.13 (d, J=6.7 Hz, 6H), 1.06 (s, 9H), 1.01 (s, 9H), 0.91 (s, 9H), 0.16 (s, 3H), 0.11 (s, 3H).

Step 4. N-(9-((2R,3R,4S,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

HF-Pyridine (26.6 g, 188 mmol) at 0° C. was diluted with pyridine (29 mL). The resulting solution was added slowly to a stirred suspension of N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (29.3 g, 47.0 mmol) in CH₂Cl₂ (290 mL) at 0° C. It was stirred at 0° C. for 2h. The reaction mixture was diluted with CH₂Cl₂ (500 mL). It was washed with water (500 mL) and sat aq NaHCO₃ (500 mL). The organic layer was dried (Na₂SO₄) and concentrated to give the product. LCMS (ES, m/z): 484.4 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.05 (s, 1H), 11.73 (s, 1H), 8.43 (s, 1H), 5.89 (d, J=7.9 Hz, 1H), 5.34 (d, J=3.9 Hz, 1H), 5.27 (t, J=5.6 Hz, 1H), 4.60 (dd, J=8.1, 3.2 Hz, 1H), 4.19-4.17 (m, 1H), 3.80-3.74 (m, 1H), 3.66-3.60 (m, 1H), 3.30 (t, J=8.0 Hz, 1H), 2.80-2.73 (m, 1H), 1.12 (d, J=6.7 Hz, 6H), 0.68 (s, 9H), −0.06 (s, 3H), −0.29 (s, 3H).

Step 5. N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3R,4S,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (21 g, 43.4 mmol) was co-evaporated with pyridine (3×50 mL) and dissolved in pyridine (210 mL). 4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (16.2 g, 47.8 mmol) was added, and it was stirred at rt for 3h and then concentrated under reduced pressure and co-evaporated with toluene (3×50 mL). The crude was purified by silica gel chromatography eluting with 0-40% EtOAc in CH₂Cl₂ (containing 0.1% Et₃N) to give the product. LCMS (ES, m/z): 786.4 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 12.06 (s, 1H), 11.71 (s, 1H), 8.19 (s, 1H), 7.44-7.42 (m, 2H), 7.37-7.22 (m, 7H), 6.92 (d, J=8.5 Hz, 4H), 5.87 (d, J=7.2 Hz, 1H), 5.44 (d, J=4.4 Hz, 1H), 4.40 (dd, J=7.3, 3.3 Hz, 1H), 4.19 (d, J=5.9 Hz, 1H), 3.75 (s, 6H), 3.54-3.35 (m, 2H), 3.34-3.28 (m, 1H), 2.83-2.71 (m, 1H), 1.11 (d, J=6.7 Hz, 6H), 0.70 (s, 9H), −0.08 (s, 3H), −0.29 (s, 3H).

Step 6: N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-fluorotetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (22 g, 28.0 mmol) in CH₂Cl₂ (220 mL) at 0° C. was added pyridine (18.11 mL, 224 mmol) and DAST (14.79 mL, 112 mmol) dropwise. The reaction was allowed to warm to rt and stirred for 7h. It was then cooled to 0° C. and quenched by slow addition of sat aq NaHCO₃ (500 mL). More CH₂Cl₂ (500 mL) was added, and the phases were separated. The organic phase was washed with sat aq NaHCO₃ (3×200 mL) and brine (300 mL), dried (Na₂SO₄), concentrated, and purified by reverse phase (C18) chromatography eluting with 45-95% of ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 788.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.06 (s, 1H), 11.56 (s, 1H), 8.02 (s, 1H), 7.44-7.42 (m, 2H), 7.34-7.23 (m, 7H), 6.92-6.89 (m, 4H), 5.71 (d, J=4.9 Hz, 1H), 5.14 (dt, J=51.0, 5.6 Hz, 1H), 5.01-4.96 (m, 1H), 3.88-3.85 (m, 1H), 3.75 (s, 6H), 3.57 (t, J=8.8 Hz, 1H), 3.50 (dd, J=10.0, 5.3 Hz, 1H), 2.81-2.74 (m, 1H), 1.12 (d, J=6.8 Hz, 6H), 0.77 (s, 9H), 0.00 (s, 3H), −0.16 (s, 3H). F-NMR: (376 MHz, DMSO-d₆, ppm) 6-193.99 (s, 1F).

Step 7: N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of N-(9-((2R,3R,4R, SR)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy) methyl)-3-((tert-butyldimethylsilyl)oxy)-4-fluorotetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (7.2 g, 6.85 mmol) in THF (70 mL) at rt was added TBAF (1.0M in THF, 8.22 mL, 8.22 mmol) dropwise. It was stirred at rt for 30 min, the solution was concentrated, and CH₂Cl₂ (300 mL) was added. The mixture was washed with NaHCO₃ (3×200 mL) and brine (200 mL), and the organic phase was separated, dried (Na₂SO₄), concentrated and purified by reverse phase (C18) chromatography eluting with 0-95% of ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 674.3 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.02 (br, 1H), 7.85 (s, 1H), 7.44-7.41 (m, 2H), 7.36-7.24 (m, 7H), 6.94-6.90 (m, 4H), 6.33 (bs, 1H), 5.78 (d, J=2.7 Hz, 1H), 5.19 (dt, J=50.2, 4.0 Hz, 1H), 4.80-4.75 (m, 1H), 4.10-4.02 (m, 1H), 3.76 (s, 6H), 3.55 (dd, J=9.3, 5.6 Hz, 1H), 3.44 (t, J=8.9 Hz, 1H), 2.79-2.71 (m, 1H), 1.12 (d, J=6.8 Hz, 6H). F-NMR: (376 MHz, DMSO-d₆) δ −194.75 (s).

Preparation 18: N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: (3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of (6aR,9R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl 2,4-dimethoxybenzoate (60 g, 108 mmol) in THF (500 mL) were added AcOH (13.59 g, 226 mmol) and TBAF in THF (1 M, 226 mL, 226 mmol). After 1h, it was concentrated under reduced pressure and purified by silica gel column chromatography eluting with 0-20% EtOAc in CH₂Cl₂ to give the product. LCMS (ES, m/z): 315.1 [M+H]⁺. 1H-NMR (400 MHz, CDCl₃) δ 7.87 (d, J=8.7 Hz, 1H), 6.58-6.46 (m, 2H), 5.51 (dt, J=5.0, 3.7 Hz, 1H), 4.31 (td, J=6.9, 3.7 Hz, 1H), 3.88 (d, J=12.0 Hz, 8H), 3.58 (dt, J=7.1, 4.7 Hz, 1H), 3.26 (dd, J=12.2, 5.0 Hz, 1H), 3.15-3.01 (m, 2H), 2.31 (s, 1H).

Step 2: (3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of (3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (33 g, 105 mmol) in pyridine (300 mL) was added tert-butylchlorodiphenylsilane (43.3 g, 157 mmol). It was stirred at rt for 4h. Then, water (300 mL) was added. Layers were separated, and the aq layer was extracted with CH₂Cl₂ (3×300 mL). The combined organic layer was washed with brine (300 mL), dried (Na₂SO₄), concentrated, and purified by silica gel column chromatography eluting with 1 to 10% EtOAc in petroleum ether to give the product. LCMS (ES, m/z): 575.3 [M+Na]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J=8.7 Hz, 1H), 7.68 (t, J=5.5 Hz, 5H), 7.54-7.38 (m, 6H), 6.69-6.60 (m, 2H), 5.40 (d, J=5.6 Hz, 1H), 5.35-5.29 (m, 1H), 4.12 (s, 1H), 4.05 (d, J=7.3 Hz, 1H), 3.85 (d, J=7.2 Hz, 6H), 3.78-3.66 (m, 1H), 3.55 (d, J=7.2 Hz, 1H), 3.14 (dd, J=11.0, 5.0 Hz, 1H), 2.86-2.77 (m, 1H), 1.03 (s, 9H).

Step 3: (3R,4S,5R)-4-((1H-imidazole-1-carbonothioyl)oxy)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of (3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (48 g, 87 mmol) in dichloroethane (500 mL) was added di(1H-imidazol-1-yl)methanethione (20.12 g, 113 mmol). It was heated at 85° C. under Ar for 1h. Then, it was concentrated and used in the next step without purification. LCMS (ES, m/z): 663.2 [M+H]⁺.

Step 4: (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of (3R,4S,5R)-4-((1H-imidazole-1-carbonothioyl)oxy)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (crude, 57.6 g, 87 mmol) in THF (40 mL) and toluene (200 mL) was added tributylstannane (139 g, 478 mmol). It was heated at 95° C., and 2,2′-(diazene-1,2-diyl)bis(2-methylpropanenitrile) (1.427 g, 8.69 mmol) in toluene (200 mL) was added over 30 min. After 1h, the resulting mixture was concentrated and purified by silica gel column chromatography eluting with 0 to 10% EtOAc in petroleum ether to give the product. LCMS (ES, m/z): 537.3 [M+H]⁺. ¹H-NMR (400 MHz, CDCl₃) δ 7.88 (d, J=8.6 Hz, 1H), 7.77-7.67 (m, 4H), 7.50-7.36 (m, 6H), 6.58-6.48 (m, 2H), 5.70 (p, J=3.8 Hz, 1H), 3.95-3.74 (m, 10H), 3.28 (dd, J=12.0, 4.6 Hz, 1H), 3.10-3.02 (m, 1H), 2.49-2.39 (m, 1H), 1.92 (ddd, J=13.3, 8.6, 4.1 Hz, 1H), 1.09 (s, 9H).

Step 5: (JR, 3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-1-oxidotetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of Ti(OiPr)₄ (23.29 mL, 78 mmol) in CH₂Cl₂ (130 mL) under Ar was added diethyl (L)-tartrate (38.3 mL, 224 mmol) dropwise. After 10 min, the mixture was cooled to −20° C., and then TBHP in decane (˜5.5M, 27.1 mL, 149 mmol) was added dropwise. After 5 min, a solution of (3R,5 S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (40 g, 74.5 mmol) in CH₂Cl₂ (130 mL) was added to the reaction at −20° C. The resulting mixture was stirred at −20° C. for 16h. It was quenched by the addition of ice water (300 mL) and allowed to warm up to rt. The precipitate was filtered off and washed with EtOAc (3×300 mL). The filtrate was washed with water (3×200 mL). The aq layer was extracted with EtOAc (400 mL). The combined organic layer was dried (Na₂SO₄), concentrated and purified by flash chromatography eluting with 0 to 70% EtOAc in petroleum ether to give the product (mixture of two isomers). LCMS (ES, m/z): 553.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.81 (d, J=8.7 Hz, 1H), 7.75-7.61 (m, 4H), 7.54-7.39 (m, 6H), 6.67-6.56 (m, 2H), 5.66 (q, J=3.8 Hz, 1H), 4.12 (dt, J=10.5, 4.4 Hz, 1H), 3.92-3.79 (m, 8H), 3.58-3.42 (m, 1H), 3.20-3.09 (m, 1H), 2.97 (d, J=15.0 Hz, 1H), 2.05 (ddd, J=14.5, 10.5, 4.3 Hz, 1H), 1.02 (s, 9H).

Step 6: (3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

A solution of (3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)-tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (21 g, 34.2 mmol) in acetic anhydride (210 mL) was heated at 110° C. After stirring of 3.5h, the reaction mixture was cooled to rt and concentrated. The residue was purified by silica gel column chromatography eluting with 0% to 20% EtOAc in petroleum ether to give the product. LCMS (ES, m/z): 535.3 [M-OAc]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.76-7.61 (m, 5H), 7.46 (dq, J=7.6, 4.5, 3.7 Hz, 6H), 6.68-6.58 (m, 2H), 6.22 (d, J=4.3 Hz, 0.65H), 5.94 (s, 0.28H), 5.51-5.46 (m, 0.33H), 5.38 (ddd, J=11.1, 7.2, 4.3 Hz, 0.65H), 3.96-3.61 (m, 9H), 2.40-2.21 (m, 2H), 2.03 (d, J=1.6 Hz, 3H), 1.01 (s, 9H).

Step 7: (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of N-(6-chloro-9H-purin-2-yl)isobutyramide (10.27 g, 42.9 mmol) in toluene (600 mL) at 0° C. was added trimethylsilyl N-(trimethylsilyl)acetimidate (23.26 g, 114 mmol). It was heated at 80° C. for 1h and was cooled to 0° C. again. To the reaction was then added a solution of (3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)-tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (17 g, 28.6 mmol) in toluene (600 mL) and trimethylsilyl trifluoromethanesulfonate (19.06 g, 86 mmol). It was heated to 80° C. and stirred under Ar for 12h. At that time, the reaction was cooled to rt, and sat aq NaHCO₃ (400 mL) was added. Layers were separated, and the aq layer was extracted with EtOAc (4×1000 mL). The combined organic phase was dried (Na₂SO₄), concentrated and purified by silica gel column chromatography eluting with 10% to 40% EtOAc in petroleum ether to give the product (mixture of a and 3 isomers). LCMS (ES, m/z): 774.3 [M+H]⁺. ¹H-NMR (400 MHz, CDCl₃) δ 8.48 (s, 0.32H), 8.35 (s, 0.69H), 7.97-7.83 (m, 1.67H), 7.70 (dq, J=8.4, 1.5 Hz, 4H), 7.54 (d, J=8.7 Hz, 0.33H), 7.49-7.36 (m, 6H), 6.57-6.36 (m, 2.5H), 6.18 (d, J=2.4 Hz, 0.7H), 5.87-5.80 (m, 0.35H), 5.73 (q, J=3.3 Hz, 0.75H), 4.23-4.01 (m, 1.2H), 3.98-3.74 (m, 7.8H), 3.11 (s, 0.73H), 2.95 (s, 0.37H), 2.57-2.36 (m, 1.75H), 2.30-2.20 (m, 0.34H), 1.23 (d, J=6.8 Hz, 2.19H), 1.19 (dd, J=6.8, 3.5 Hz, 4.38H), 1.09 (d, J=1.7 Hz, 9H).

Step 8. (2R,3R,5S)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)-5-(hydroxymethyl)-tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate

To a solution of (3R,5 S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (20 g, 25.8 mmol) in THF (135 mL) was added TBAF in THF (1 M, 31 mL, 31 mmol) dropwise. After 1h, the reaction mixture was concentrated and purified by column chromatography using 1% to 10% MeOH in CH₂Cl₂ as the eluent to give a mixture of two isomers. It was re-purified by reverse phase (C18) chromatography eluting with 10 to 45% ACN in aq NH₄CO₃ (5 mM) to give the product. LCMS (ES, m/z): 536.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 8.83 (s, 1H), 7.72 (d, J=8.7 Hz, 1H), 6.65-6.55 (m, 2H), 6.18 (d, J=2.5 Hz, 1H), 5.80 (q, J=3.5 Hz, 1H), 5.22 (t, J=5.1 Hz, 1H), 3.93-3.67 (m, 9H), 2.85 (p, J=6.9 Hz, 1H), 2.70 (ddd, J=13.4, 8.5, 4.5 Hz, 1H), 2.36 (dt, J=14.1, 5.0 Hz, 1H), 1.06 (dd, J=6.8, 3.3 Hz, 6H).

Step 9: 2-amino-9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one

To a solution of (2R,3R,5 S)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrothiophen-3-yl 2,4-dimethoxybenzoate (6.0 g, 11.19 mmol) in MeOH (300 mL) were added 2-mercaptoethanol (3.50 g, 44.8 mmol) and NaOMe (10.08 g, 56 mmol, 30% in Me0H). It was heated at 60° C. for 16h, cooled to rt, and conc. HCl (4 mL) was added. The resulting mixture was concentrated, and water (100 mL) and EtOAc (100 mL) were added. Layers were separated, and the aq layer was extracted with EtOAc (3×100 mL). The aq layer was basified with NaHCO₃ (solid) to ˜pH 8 and stirred at rt for 1h. The precipitate was filtered and kept under reduced pressure to give the product. LCMS (ES, m/z): 284.1 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 7.98 (s, 1H), 6.46 (s, 2H), 5.57 (dd, J=10.9, 3.9 Hz, 2H), 5.12 (t, J=5.4 Hz, 1H), 4.48 (p, J=4.1 Hz, 1H), 3.78-3.53 (m, 3H), 2.11-1.99 (m, 2H).

Step 10: N-(9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3R,5 S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one (580 mg, 2.047 mmol) was co-evaporated with pyridine (3×20 mL) and then re-dissolved in pyridine (20 mL). The mixture was cooled to 0° C. and then treated with chlorotrimethylsilane (1557 mg, 14.33 mmol). It was warmed to rt and stirred for 2h. Then, the reaction was cooled to 0° C. again, and isobutyric anhydride (486 mg, 3.07 mmol) was added dropwise. It was warmed to rt and stirred for 2h. The reaction was quenched by the addition of methanol (5 mL). After 5 min, NH₄OH (ca 29%, 10 mL) was added. The mixture was stirred at rt for 30 min. Then, it was concentrated and purified by column chromatography on silica gel eluting with 10% to 20% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 354.1 [M+H]⁺. ¹H-NMR (300 MHz, CD₃OD) δ: 8.40 (s, 1H), 5.83-5.82 (m, 1H), 4.62-4.59 (m, 1H), 3.89-3.80 (m, 2H), 3.79-3.74 (m, 1H), 2.73-2.64 (m, 1H), 2.19-2.11 (m, 2H), 1.20 (d, J=6.8 Hz, 6H).

Step 11: N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3R,5 S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (510 mg, 1.443 mmol) was co-evaporated with pyridine (3×5 mL) and then re-suspended in pyridine (7 mL). To the suspension was added 4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (538 mg, 1.587 mmol), and the mixture was stirred at rt for 2h. At that time the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel eluting with 1% to 4% MeOH in CH₂Cl₂ (containing 1% Et₃N) to give the product. LCMS (ES, m/z): 656.0 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD) δ: 8.02 (s, 1H), 7.51-7.43 (m, 2H), 7.40-7.17 (m, 7H), 6.91-6.82 (m, 4H), 5.85-5.84 (d, J=2.3 Hz, 1H), 4.59-4.57 (m, 1H), 4.02-3.95 (m, 1H), 3.78 (s, 6H), 3.52-3.34 (m, 3H), 2.72-2.68 (m, 1H), 1.95-191 (m, 1H), 1.38 (d, J=6.8 Hz, 6H).

Preparation 19: N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: ((2R,3R,4S)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate

To a stirred solution of ((2R,3R,4S)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methyl benzoate (20.0 g, 49.7 mmol) in dry 1,2-dichloroethane (200 mL) was added tri-N-butyltin methoxide (28.8 mL, 99 mmol). The resulting mixture was stirred at 80° C. for 3h and then concentrated in vacuo. The residue was diluted in 500 mL of ethyl acetate and washed with sat aq. NH₄Cl (500 mL) and brine (500 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-40% EtOAc/Hexane. LCMS (ES, m/z): 343.2 [M+H-H₂O]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.18-8.00 (m, 7H), 7.68-7.57 (m, 3H), 7.61-7.35 (m, 7H), 5.73 (dd, J=8.3, 4.6 Hz, 1H), 5.65 (dt, J=3.9, 2.0 Hz, 1H), 5.54 (t, J=4.7 Hz, 0H), 5.50-5.40 (m, 2H), 5.36-5.24 (m, 1H), 4.87 (dtd, J=25.5, 4.0, 1.5 Hz, 1H), 4.74-4.46 (m, 4H), 4.15 (q, J=7.2 Hz, 1H), 3.39 (d, J=4.1 Hz, 1H), 3.30 (dd, J=8.6, 3.4 Hz, 1H), 2.07 (s, 2H), 1.41-1.23 (m, 3H).

Step 2. (2R,3R,4S)-3-fluoro-2-hydroxy-5-(methoxyimino)pentane-1,4-diyl dibenzoate

To a stirred solution of ((2R,3R,4S)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate (17.9 g, 49.7 mmol) in dry MeOH (100 mL) was added O-methylhydroxylamine hydrochloride (6.23 g, 74.6 mmol), followed by triethylamine (10.39 mL, 74.6 mmol). The reaction mixture was stirred at rt for 2h and then concentrated in vacuo. The residue was diluted in 500 mL of ethyl acetate and washed with sat aq. NH₄Cl (500 mL) and brine (500 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was used for the next step directly without further purification. LCMS (ES, m/z): 390.2 [M+H]⁺.

Step 3: (2R,3S,4S)-3-fluoro-5-(methoxyimino)-2-(((4-nitrophenyl)sulfonyl)oxy)pentane-1,4-diyl dibenzoate

To a stirred solution of (2R,3R,4S)-3-fluoro-2-hydroxy-5-(methoxyimino)pentane-1,4-diyl dibenzoate (19.4 g, 49.7 mmol) in dry EtOAc (100 mL) was added 4-nitrobenzenesulfonyl chloride (16.5 g, 74.6 mmol), followed by triethylamine (10.4 mL, 74.6 mmol). The reaction mixture was stirred at rt for 18h and was then diluted with 200 mL of ethyl acetate, washed with water (300 mL) and brine (300 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-30% EtOAc/Hexane. LCMS (ES, m/z): 575.3 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.25-7.99 (m, 7H), 7.94-7.80 (m, 2H), 7.68-7.55 (m, 2H), 7.57-7.46 (m, 3H), 7.50-7.38 (m, 3H), 6.55 (ddd, J=17.2, 5.3, 2.8 Hz, 0H), 5.89 (ddd, J=17.7, 6.2, 4.1 Hz, 1H), 5.46-5.37 (m, 1H), 5.40-5.28 (m, 1H), 5.21 (t, J=4.3 Hz, 0H), 4.84 (tdd, J=12.8, 2.6, 1.5 Hz, 1H), 4.53 (dddd, J=23.1, 13.0, 7.0, 1.8 Hz, 1H), 4.14 (q, J=7.2 Hz, 1H), 3.99 (d, J=36.3 Hz, 4H), 2.06 (s, 2H), 1.44-1.23 (m, 2H).

Step 4. (2S,3S,4S)-2-bromo-3-fluoro-5-(methoxyimino)pentane-1,4-diyl dibenzoate

To a stirred solution of (2R,3 S,4S)-3-fluoro-5-(methoxyimino)-2-(((4-nitrophenyl)sulfonyl)oxy)pentane-1,4-diyl dibenzoate (21.3 g, 37.1 mmol) in dry DMF (100 mL) was added freshly opened lithium bromide powder (16.1 g, 185 mmol). The resulting mixture was stirred at 60° C. for 18h. The reaction mixture was diluted in 300 mL of ethyl acetate and washed with water (500 mL) and brine (500 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-25% EtOAc/Hexane to give product. LCMS (ES, m/z): 452.1 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.15-8.03 (m, 11H), 7.69-7.56 (m, 8H), 7.56-7.41 (m, 12H), 6.93-6.86 (m, 1H), 6.45 (ddd, J=14.7, 5.9, 4.7 Hz, 0H), 5.93 (ddd, J=11.8, 6.7, 5.9 Hz, 2H), 5.32 (s, 1H), 5.29-5.10 (m, 3H), 4.89-4.77 (m, 3H), 4.80-4.67 (m, 3H), 4.60-4.40 (m, 3H), 3.95 (d, J=16.9 Hz, 2H), 3.89 (s, 6H), 1.32-1.22 (m, 1H).

Step 5. (2S,3S,4S)-2-bromo-3-fluoro-5-oxopentane-1,4-diyl dibenzoate

To a stirred solution of (2S,3S,4S)-2-bromo-3-fluoro-5-(methoxyimino)pentane-1,4-diyl dibenzoate (20.0 g, 44.2 mmol) in THF (200 mL) was added 37% aqueous solution of formaldehyde (32.9 mL, 442 mmol) and IN HCl (44.2 mL, 44.2 mmol). The resulting mixture was stirred at 55° C. for 5h. The reaction mixture was concentrated in vacuo to remove most of the THF. The residue was diluted in 300 mL of ethyl acetate and washed with water (300 mL) and brine (300 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was used for the next step without further purification. LCMS (ES, m/z): 423.2 [M+H]⁺.

Step 6: ((2R,3S,4R)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrothiophen-2-yl)methyl benzoate

To a stirred solution of (2S,3S,4S)-2-bromo-3-fluoro-5-oxopentane-1,4-diyl dibenzoate (18.7 g, 44.2 mmol) in NMP (150 mL) at 0° C. was added sodium hydrosulfide (5.0 g, 89.0 mmol). The resulting mixture was stirred at 0° C. for 30 min and then at rt for 30 min. The reaction mixture was diluted in 300 mL of ethyl acetate and washed with water (300 mL) and brine (300 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was used for the next step without further purification. LCMS (ES, m/z): 359.2 [M+H—H₂O]⁺.

Step 6: ((2R,3S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methyl benzoate

To a stirred solution of ((2R,3 S,4R)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrothiophen-2-yl)methyl benzoate (16.6 g, 44.2 mmol) in dry THF (150 mL) at 0° C. was added acetic anhydride (8.3 mL, 133 mmol) and trimethylamine (18.5 mL, 133 mmol). The resulting mixture was stirred at 0° C. for 30 min and then at rt for 2h. The reaction was quenched by addition of MeOH, and the reaction mixture was concentrated in vacuo to remove most of the THF. The residue was diluted in 300 mL of ethyl acetate and washed with water (300 mL) and brine (300 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-25% EtOAc/Hexane to give product. LCMS (ES, m/z): 441.2 [M+Na]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.15-8.02 (m, 4H), 7.68-7.57 (m, 2H), 7.55-7.42 (m, 4H), 6.02 (t, J=2.4 Hz, 1H), 5.88 (ddd, J=6.1, 3.6, 2.4 Hz, 1H), 5.50-5.42 (m, 0H), 5.35 (dd, J=7.3, 3.6 Hz, 0H), 5.32 (s, 0H), 4.80-4.69 (m, 1H), 4.56 (dd, J=11.6, 5.9 Hz, 1H), 4.26-4.10 (m, 2H), 2.25-2.11 (m, 1H), 2.08 (d, J=10.0 Hz, 3H), 1.29 (t, J=7.1 Hz, 1H).

Step 7: ((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-2-yl)methyl benzoate

To a suspension of N-(6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (15.86 g, 71.7 mmol) in ClCH₂CH₂Cl (300 mL) was added (Z)-trimethylsilyl N-(trimethylsilyl)acetimidate (35.1 mL, 143 mmol). The suspension was stirred at 70° C. overnight and was then cooled to −15° C. To this mixture was added ((2R,3S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methyl benzoate (10 g, 23.90 mmol), followed by TMS-OTf (8.64 mL, 47.8 mmol). The reaction mixture was stirred at −15° C. for 2h, then at rt for 5h and finally at 70° C. for 5d. The reaction mixture was allowed to cool to RT and was then filtered. The filtrate was washed with sat. aq. NaHCO₃, brine and then dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by silica gel column, eluting with 0-60% EtOAc/Hexane. Subsequent recrystallization from EtOAc gave product. LCMS (ES, m/z): 580.5 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 12.19 (s, 0H), 9.56 (s, 1H), 8.12-8.06 (m, 1H), 8.02-7.96 (m, 1H), 7.66 (s, 0H), 7.71-7.58 (m, 1H), 7.57-7.50 (m, 1H), 7.54-7.42 (m, 1H), 6.59 (ddd, J=25.3, 7.4, 3.1 Hz, 1H), 6.20 (d, J=7.4 Hz, 0H), 5.68-5.59 (m, 1H), 4.87 (ddd, J=11.7, 7.7, 1.5 Hz, 1H), 4.26-4.11 (m, 1H), 2.96 (hept, J=6.8 Hz, 1H), 1.38 (dd, J=19.0, 6.9 Hz, 3H).

Step 8: N-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of ((2R,3 S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-2-yl)methyl benzoate (0.96 g, 1.66 mmol) in THF (5 mL)/MeOH (4 mL)/H₂O (1 mL) at 0° C. was added 2N sodium hydroxide (1.8 mL, 3.6 mmol). The reaction mixture was stirred at 0° C. for 30 min and then neutralized with acetic acid (0.38 mL, 6.6 mmol). The product was collected by filtration and carried on to the next step without further purification. LCMS (ES, m/z): 372.3 [M+H]⁺.

Step 9: N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of N-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (0.62 g, 1.66 mmol) in pyridine (25 mL) at 0° C. was added 4,4′-dimethoxytrityl chloride (0.84 g, 2.48 mmol). The reaction mixture was stirred at 0° C. for 3h. The reaction was quenched with H₂O (1 mL), and the mixture was concentrated. The residue was diluted in 100 mL of ethyl acetate and washed with saturated aq. NaHCO₃ (100 mL) and brine (100 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-100% EtOAc/Hexane containing 0.1% Et₃N to give product. LCMS (ES, m/z): 674.6 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 11.96 (s, 0H), 8.35 (s, 0H), 7.70 (s, 0H), 7.63-7.56 (m, 1H), 7.50-7.43 (m, 2H), 7.38-7.24 (m, 1H), 6.93-6.85 (m, 2H), 5.92 (d, J=8.3 Hz, 0H), 5.35-5.23 (m, 1H), 4.15 (q, J=7.1 Hz, 1H), 3.85-3.69 (m, 3H), 3.46 (dd, J=10.2, 5.4 Hz, 0H), 3.35 (dd, J=10.2, 5.2 Hz, 0H), 2.09 (d, J=19.4 Hz, 2H), 1.34-1.22 (m, 2H), 1.01 (d, J=6.8 Hz, 1H), 0.91 (d, J=6.9 Hz, 1H).

Preparation 20: N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1: N-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a suspension of 5-amino-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (5.0 g, 32.9 mmol) in anhydrous DMF (60 mL) was added isobutyric anhydride (12.5 mL, 76.0 mmol) dropwise. The reaction mixture was refluxed at 150° C. for 1h. The reaction was quenched with MeOH (6.6 mL, 164 mmol) and concentrated in vacuo. The residue was taken up in DCM (50 mL)/Hexane (100 mL) and was stirred at vigorously at rt for 15 min. The product was collected by filtration. LCMS (ES, m/z): 223.2 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 16.04 (s, 1H), 12.19 (s, 1H), 11.78 (s, 1H), 2.78 (hept, J=6.7 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H).

Step 2: ((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophen-2-yl)methyl benzoate

To a mixture of ((2R,3 S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methyl benzoate (1.5 g, 3.6 mmol) and N-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide (0.96 g, 4.3 mmol) in nitromethane (20 mL) was added BF₃*Et₂O (0.54 mL, 4.3 mmol) and the resulting mixture was heated at 100° C. under microwave irradiation for 1h. The reaction mixture was cooled, diluted in 100 mL of ethyl acetate and washed with saturated aq. NaHCO₃ (100 mL) and brine (100 mL). The organic portion was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-35% EtOAc/Hexane. LCMS (ES, m/z): 581.4 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 12.27 (s, 2H), 9.73 (s, 2H), 8.19-7.93 (m, 8H), 7.86-7.80 (m, 0H), 7.71-7.55 (m, 4H), 7.57-7.39 (m, 8H), 7.40-7.33 (m, 0H), 6.76 (d, J=6.7 Hz, 2H), 6.62-6.48 (m, 2H), 5.79 (t, J=2.9 Hz, 1H), 5.69 (t, J=2.9 Hz, 1H), 5.62-5.54 (m, 2H), 4.89 (ddd, J=11.6, 7.8, 1.3 Hz, 2H), 4.78-4.65 (m, 1H), 4.31-4.18 (m, 2H), 4.15 (q, J=7.1 Hz, 3H), 2.95 (hept, J=6.9 Hz, 2H), 2.81-2.68 (m, 1H), 2.07 (s, 4H), 1.39 (dd, J=17.5, 6.9 Hz, 10H), 1.34-1.24 (m, 8H).

Step 3: N-(3-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of ((2R,3 S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophen-2-yl)methyl benzoate (4.5 g, 7.8 mmol) in THF (35 mL)/MeOH (28 mL)/H (7 mL) at 0° C. was added 2N sodium hydroxide (8.6 mL, 17.2 mmol). The reaction mixture was stirred at 0° C. for 1h and then neutralized with acetic acid (2.3 mL, 39.0 mmol). Product was collected by filtration and carried on to the next step without further purification. LCMS (ES, m/z): 373.3 [M+H]⁺.

Step 4: N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of N-(3-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide (1.8 g, 4.8 mmol) in pyridine (30 mL) at 0° C. was added 4,4′-dimethoxytrityl chloride (1.8 g, 5.3 mmol). The reaction mixture was stirred at 0° C. for 1h. The reaction was quenched with H₂O (1 mL), and the mixture was concentrated. The residue was diluted in 100 mL of ethyl acetate and washed with saturated aq. NaHCO₃ (100 mL) and brine (100 mL). The separated organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by a silica gel column, eluting with 0-100% EtOAc/Hexane containing 0.1% Et₃N to give product. LCMS (ES, m/z): 675.5 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 12.16 (s, 1H), 8.48 (s, 0H), 7.57-7.50 (m, 1H), 7.49-7.37 (m, 2H), 7.39-7.29 (m, 0H), 7.31 (s, 1H), 7.32-7.14 (m, 1H), 6.91-6.81 (m, 2H), 6.22-6.16 (m, 0H), 5.42-5.31 (m, 1H), 3.77 (d, J=36.0 Hz, 7H), 3.46 (dd, J=10.2, 5.6 Hz, 0H), 3.36 (dd, J=10.2, 5.4 Hz, 0H), 2.17 (p, J=6.9 Hz, 0H), 1.06 (dd, J=26.1, 6.9 Hz, 3H).

Preparation 21: N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1: (2R,3S,4R,5R)-2-((benzoyloxy)methyl)-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophene-3,4-diyl dibenzoate

BF₃.OEt₂ (3.65 mL, 28.8 mmol) was added dropwise to a mixture of (2R,3R,4S,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrothiophene-3,4-diyl dibenzoate (10.0 g, 19.2 mmol) and N-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5 yl)isobutyramide (5.98 g, 26.9 mmol) in MeNO₂ (180 mL) at ambient temperature. Upon completion of addition, the mixture was heated at 120° C. in a microwave reactor for 45 min. The sample was cooled to rt and concentrated in vacuo. The residue was purified by flash column chromatography eluting with EtOAc/isohexane (10-90%) to give desired product. LCMS (ES, m/z): 683.5 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 12.24 (s, 1H), 9.77 (s, 1H), 8.08-7.95 (m, 4H), 7.94-7.87 (m, 2H), 7.70-7.35 (m, 9H), 6.84 (d, J=5.9 Hz, 1H), 6.67 (dd, J=5.9, 3.9 Hz, 1H), 6.49 (t, J=3.7 Hz, 1H), 5.52 (dd, J=11.4, 7.8 Hz, 1H), 5.04 (dd, J=11.4, 7.8 Hz, 1H), 4.28 (m, 1H), 2.97 (hept, J=6.9 Hz, 1H), 1.42 (dd, 6.9 Hz, 6H).

Step 2: N-(3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a stirred solution of the product from Step 1 (5.3 g, 7.8 mmol) dissolved in Pyridine (8 mL) and MeOH (32 mL) at 25° C. was added sodium hydroxide (1.24 g, 31.1 mmol) in one portion. The mixture was stirred at 25° C. for 15 min before the addition of acetic acid (1.8 mL, 31.1 mmol). The mixture was concentrated in vacuo, and the residue was purified by flash column chromatography eluting with EtOAc/isohexane (70-100%) to give the desired product. LCMS (ES, m/z): 371.3 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 12.24 (s, 1H), 11.96 (s, 1H), 5.91 (d, J=5.6 Hz, 1H), 5.73 (d, J=5.5 Hz, 1H), 5.43 (d, J=5.0 Hz, 1H), 5.12 (m, 1H), 4.81 (m, br, 1H), 4.40 (m, 1H), 3.89-3.77 (m, 1H), 3.53 (m, 1H), 3.46-3.36 (m, 1H), 2.79 (m, 1H), 1.14 (dd, J=6.7, 1.2 Hz, 6H).

Step 3: N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To the product from step 2 (2.3 g, 6.2 mmol) was added pyridine (62 mL) at ambient temperature. To this mixture was added DMTrCl (2.3 g, 6.8 mmol). After 1h, water (2 mL) was added, and it was concentrated in vacuo. Ethyl acetate (15 mL), water (5 mL) and brine (1 mL) were added. Layers were separated, and the aqueous layer was extracted with ethyl acetate twice (20 mL×2). The combined organics were dried over MgSO₄, concentrated in vacuo and purified by flash column chromatography, eluting with 0 to 80% EtOAc in Hexane to give the desired product. LCMS (ES, m/z): 673.4 [M+H]⁺ 0.1H NMR (500 MHz, DMSO-d₆) δ 12.24 (s, 1H), 11.96 (s, 1H), 7.50-7.36 (m, 2H), 7.35-7.10 (m, 9H), 6.96-6.82 (m, 4H), 5.88 (d, J=4.1 Hz, 1H), 5.83 (d, J=5.1 Hz, 1H), 5.42 (d, J=5.7 Hz, 1H), 4.66 (q, J=4.1 Hz, 1H), 4.45 (td, J=5.9, 3.5 Hz, 1H), 3.81-3.69 (6H), 3.65 (ddd, J=8.4, 6.0, 4.3 Hz, 1H), 3.46-3.35 (m, 1H), 3.19 (dd, J=9.4, 7.9 Hz, 1H), 2.78 (h, J=6.8 Hz, 1H), 1.22-1.05 (m, 6H).

Preparation 22: ammonium (2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

Step 1: N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one (1.7 g, 5.7 mmol) was co-evaporated with pyridine (3×5 mL) and then, re-dissolved in pyridine (34 mL). To the mixture at 0° C. was added chlorotrimethylsilane (4.32 g, 39.8 mmol) dropwise. It was stirred at rt for 1h and then, cooled to 0° C. again. Isobutyric anhydride (1.348 g, 8.52 mmol) was added dropwise, and it was stirred at rt for 3h. It was quenched by the addition of water (8.5 mL). After 5 min, NH₄OH (ca. 29%, 17 mL) was added, and the mixture was stirred for 30 min. It was concentrated and purified by column chromatography eluted with 1 to 30% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 396.9 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 9.52 (br s, 2H), 8.39 (s, 1H), 5.79 (d, J=7.1 Hz, 1H), 5.59 (s, 1H), 5.40 (s, 1H), 5.22 (s, 1H), 4.55 (d, J=6.7 Hz, 1H), 4.21 (s, 1H), 3.77 (t, J=9.3 Hz, 1H), 3.61 (s, 1H), 3.30 (dt, J=6.4, 3.3 Hz, 1H), 2.78 (p, J=6.9 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H).

Step 2: N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a mixture of N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (480 mg, 1.299 mmol) in pyridine (10 mL) was added 4,4′-(chloro(phenyl)methylene)-bis(methoxybenzene) (484 mg, 1.43 mmol). It was stirred at rt for 16h and then, concentrated. The crude was purified by column chromatography on silica gel eluted with 1 to 30% MeOH in CH₂Cl₂ (containing 1% Et₃N) to give the product. LCMS (ES, m/z): 672.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆+D₂O): δ 8.08 (s, 1H), 7.39 (d, J=7.2 Hz, 2H), 7.32 (t, J=7.6 Hz, 2H), 7.26 (dt, J=9.1, 3.3 Hz, 5H), 6.94-6.87 (m, 4H), 5.75 (d, J=5.9 Hz, 1H), 4.39 (dd, J=5.9, 3.5 Hz, 1H), 4.14 (t, J=3.9 Hz, 1H), 3.74 (s, 6H), 3.49-3.37 (m, 2H), 3.33 (dd, J=14.5, 7.3 Hz, 1H), 2.87-2.67 (m, 1H), 1.11 (dd, J=6.8, 1.6 Hz, 6H).

Step 3: N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide and N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (580 mg, 0.863 mmol) in DMF (5 mL) at rt was added 1H-imidazole (147 mg, 2.16 mmol) and tert-butylchlorodimethylsilane (156 mg, 1.04 mmol). After 6h, the mixture was diluted with EtOAc (50 mL) and washed with sat aq NaHCO₃ (2×20 mL) and brine (20 mL). It was dried (Na₂SO₄), concentrated, and purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in water to give the products. LCMS (ES, m/z): 786.3 [M+H]⁺.

Step 4. (2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phenyl phosphonate and (2R,3S,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phenyl phosphonate

To a solution of a mixture of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide and N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl) (phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (220 mg, 0.280 mmol) in pyridine (2 mL) at 0° C. was added diphenyl phosphonate (98 mg, 0.420 mmol). The resulting mixture was stirred at rt for 20 min. It was used in the next reaction step without purification. LCMS (ES, m/z): 926.2 [M+H]⁺.

Step 5. ammonium (2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

To the reaction mixture from Step 4 at 0° C. was added Et₃N (0.28 mL, 2.0 mmol) and water (0.28 mL). It was stirred at rt for 30 min. It was concentrated, and the residue was partitioned between CH₂Cl₂ (40 mL) and aq NaHCO₃ (5%, 30 mL). The organic layer was washed with aq NaHCO₃ (5%, 2×30 mL), dried (Na₂SO₄), concentrated and purified by silica gel column chromatography using 0-10% MeOH in CHCl₃ containing 1% Et₃N to give a mixture. The mixture was further purified by prep-HPLC Prep-HPLC (XBridge Shield RP18 OBD Column, 19×150 mm) eluted with 46 to 79% ACN in aq NH₄HCO₃ (10 mM) over 7 min to give the product. LCMS (ES, m/z): 850.2 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 8.18 (s, 1H), 7.68 (s, 0.5H), 7.59-7.49 (m, 2H), 7.45-7.36 (m, 4H), 7.37-7.30 (m, 2H), 7.28-7.22 (m, 1H), 6.95-6.87 (m, 4H), 6.16-6.07 (m, 2H), 4.88-4.87 (m, 1H), 4.69 (dd, J=7.3, 3.3 Hz, 1H), 3.81 (s, 6H), 3.51 (dd, J=4.9, 1.9 Hz, 2H), 3.37 (s, 1H), 2.67 (p, J=6.9 Hz, 1H), 1.21 (dd, J=6.9, 0.9 Hz, 6H), 0.77 (s, 9H), 0.01 (s, 3H), −0.28 (s, 3H). ³¹P-NMR (162 MHz, DMSO-d₆): δ −0.74 (s, 1P).

Preparation 23: 2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]oxy}-phosphoryl)-β-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-one

Step 1: 2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]foxy}-phosphoryl)-β-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-one

To a stirred solution of 9-(2,3-di-O-acetyl-β-D-xylofuranosyl)-2-[(2-methylpropanoyl)-amino]-1,9-dihydro-6H-purin-6-one (100 mg, 0.229 mmol) in pyridine (0.25 mL) and 1,4-dioxane (0.75 mL) was added a freshly prepared solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (50 mg, 0.247 mmol) in 1,4-dioxane (0.25 mL). The reaction mixture was stirred at ambient temperature for 10 min, and then a solution of tributylammonium pyrophosphate (189 mg, 0.344 mmol) in DMF (0.69 mL) was added, followed by addition of tributylamine (0.23 mL, 0.968 mmol) in one portion at ambient temperature. The reaction mixture was stirred for 10 min at ambient temperature, and then a solution of iodine (29.0 mg, 0.114 mmol) in pyridine (0.50 mL) and water (0.05 mL) was added. The reaction mixture was stirred for 15 min, excess iodine was quenched with 5% aq NaHSO₃ (3 mL), and the reaction mixture was evaporated to dryness. The residue was dissolved in 10 mL H₂O, and after standing at rt for 30 min, 28% aq ammonium hydroxide (5 mL) was added. The reaction mixture was stirred at 50° C. for 5h. LCMS indicated full conversion to desired product, and the mixture was filtered and lyophilyzed. The product was purified using mass-directed reverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part#186002568] using a gradient solvent system with MeCN and 100 mM aq triethylammonium acetate. Lyophilization of the product fractions furnished 2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]oxy}phosphoryl)-3-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-one as the tetra-triethylamine salt. LCMS (ES, m/z): 522 [M−H]⁻.

Preparation 24: ((2R,3S,4R,5S)-5-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

Step 1. ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol

To the stirred suspension of (2S,3R,4S,5R)-2-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (30 mg, 0.105 mmol) in 1,4-dioxane (0.3 mL) was added trimethyl orthoformate (0.22 mL, 2.011 mmol) in one portion at ambient temperature, followed by p-toluenesulfonic acid monohydrate (22 mg, 0.116 mmol). The reaction mixture was stirred at ambient temperature for 16h. LCMS indicated significant conversion to desired product, and the crude mixture was carefully quenched by adding triethylamine (0.05 mL) at 0° C. Following concentration, the residue was purified by flash column chromatography on 12 gram silica gel using a gradient solvent system with MeOH and CH₂Cl₂. Concentration of the product fractions furnished ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol. LCMS (ES, m/z): 310 [M+H]⁺.

Step 2: ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl tetrahydrogen triphosphate

To the stirred suspension of ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol (16 mg, 0.052 mmol) in pyridine (0.05 mL) and 1,4-dioxane (0.15 mL) was added DMF (0.05 mL) to form a homogeneous solution. To this solution was added a freshly prepared solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (12 mg, 0.059 mmol) in 1,4-dioxane (0.05 mL) at ambient temperature. The reaction mixture was stirred at ambient temperature for 15 min, and then a solution of tributylammonium pyrophosphate (43 mg, 0.078 mmol) in DMF (0.10 mL) was added, followed by tributylamine (0.052 mL, 0.219 mmol). The reaction mixture was stirred at ambient temperature for 15 min, and then a solution of iodine (6.58 mg, 0.026 mmol) in pyridine (0.10 mL) and water (0.01 mL) was added. The reaction mixture was stirred at ambient temperature for 15 min and excess iodine was quenched with 5% aqueous NaHSO₃ (0.5 mL). LCMS indicated significant conversion to desired product, and the reaction mixture was concentrated to yield ((3 aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl tetrahydrogen triphosphate, which was used directly in the next reaction step without additional purification. LCMS (ES, m/z): 548 [M−H]⁻.

Step 3. ((2R,3S,4R,5S)-5-(7-amino-H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

To the stirred solution of crude ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl tetrahydrogen triphosphate (49.4 mg, 0.090 mmol) in water (0.15 mL) and DMF (0.15 mL) was added formic acid (0.4 mL, 10.60 mmol) in one portion. The reaction mixture was stirred at ambient temperature for 18h. LCMS indicated significant conversion to desired product, and the mixture was filtered and lyophilyzed. The product was purified using mass-directed reverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 m, 19 mm×150 mm, [Waters Part#186002568] using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate. Lyophilization of the product fractions furnished ((2R,3 S,4R,5 S)-5-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate. LCMS (ES, m/z): 506 [M−H]⁻.

Preparation 25: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

Step 1: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

To a mixture of 2-fluoroadenosine (200 mg, 0.701 mmol) in trimethylphosphate (1.948 mL, 16.83 mmol) was added tributylamine (0.500 mL, 2.104 mmol), and the mixture was stirred 15 min at rt and then cooled in an ice/brine bath. Then POCl₃ (0.137 mL, 1.472 mmol) was added dropwise with bath temperature maintained at −5 to 0° C. After 1.25h, a 0° C. mixture of tributylammonium pyrophosphate (327 mg, 0.596 mmol), MeCN (2.8 mL) and tributylamine (1.000 mL, 4.21 mmol) were added, and the mixture was allowed to warm to rt, followed by 16h stirring at rt. The mixture was purified directly by reverse phase HPLC using a gradient of 1-20% MeCN with 100 mM aqueous triethylammonium acetate to furnish ((2R,3 S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate mg. LCMS (ES, m/z): 524 [M−H]⁻.

Preparation 26: 3′-(aminomethyl)-3′-deoxyguanosine 5′-(tetrahydrogen triphosphate)

The title compound was prepared according to published procedures (WO2015161137).

The Preparations below were used as shown or were further modified through additional synthetic manipulations analogous to those described in Preparations 1-26.

Preparation 27: 2-amino-9-((β-D-xylofuranosyl)-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures (Journal of Medicinal Chemistry 1987, 30(6), 982-991).

Preparation 28: 5-amino-3-((β-D-ribofuranosyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one

The title compound was prepared according to published procedures (Journal of Organic Chemistry 2007, 72(1), 173-179).

Preparation 29: 9-{3-azido-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-β-D-ribofuranosyl}-N-(phenylcarbonyl)-9H-purin-6-amine

The title compound was prepared according to published procedures (Bulletin of the Korean Chemical Society 2004, 25(2), 243-248 and Nucleosides, Nucleotides & Nucleic Acids 2005 24(10-12), 1707-1727).

Preparation 30: 1-(β-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine

The title compound was prepared according to published procedures (Tetrahedron 1993, 49(3), 557-570).

Preparation 31: 1-(β-D-ribofuranosyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures (Tetrahedron 1993, 49(3), 557-570)

Preparation 32: 4′-thioadenosine

The title compound was prepared according to published procedures (Journal of Medicinal Chemistry 2006, 49(5), 1624-1634).

Preparation 33: 7-((β-D-ribofuranosyl)-3,7-dihydro-4H-imidazo[4,5-d][1,2,3]triazin-4-one

The title compound was prepared according to published procedures (Organic & Biomolecular Chemistry 2014, 12(23), 3813-3815).

Preparation 34: 3-β-D-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine

The title compound was prepared according to published procedures (Biochemistry 2005, 44(37), 12445-12453).

Preparation 35: 7-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures (Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry 1995 (12), 1543-50).

Preparation 36: 1-(3-deoxy-β-D-erythro-pentofuranosyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures (Chemical & Pharmaceutical Bulletin 1996, 44(2), 288-295).

Preparation 37: 7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures (Synthesis 2006 (12), 2005-2012).

Preparation 38: (2S,3R,4S,5R)-2-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures (WO2015148746).

Preparation 39: N-(8-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4-yl)benzamide

The title compound was prepared according to published procedures (WO2015148746).

Preparation 40: 7-(2-deoxy-4-ethynyl-β-D-erythro-pentofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures (WO2015148746).

Preparation 41: 9-(2-chloro-2-deoxy-β-D-arabinofuranosyl)-9H-purin-6-amine

The title compound was prepared according to published procedures (Journal of the American Chemical Society 1996, 118(46), 11341-11348).

Preparation 42: 2′-deoxy-2′-methyladenosine

The title compound was prepared according to published procedures (Synthesis 2005 (17), 2865-2870).

Preparation 43: (2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((R)-1-hydroxyethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures (Bioorganicheskaya Khimiya 1989, 15(7), 969-975).

Preparation 44: 5-fluoro-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures (Nucleosides, Nucleotides, & Nucleic Acids 2004, 23(1-2), 161-170).

Preparation 45: 1-(β-D-ribofuranosyl)-1,5-dihydro-4H-imidazo[4,5-d ]pyridazin-4-one

The title compound was prepared according to published procedures (Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999), 1989 (10), 1769-1774).

Preparation 46: 2-amino-9-[(2R,3R,4S,5S)-5-fluoro-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures (WO2014099941)

Preparation 47: 2-amino-9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)-4-methylenetetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures (Journal of Medicinal Chemistry 1992, 35, 2283-2293).

Preparation 48: (2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures (Tetrahedron Letters 1994, 35(30), 5339).

The following experimental procedures detail the preparation of specific examples of the instant disclosure. The compounds of the examples are drawn in their neutral forms in the procedures and tables below. In some cases, the compounds were isolated as salts depending on the method used for their final purification and/or intrinsic molecular properties. The examples are for illustrative purposes only and are not intended to limit the scope of the instant disclosure in any way.

EXAMPLES Example 1: (5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][11,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide

Step 1: (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) phosphonate

To a solution of (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (3 g, 3.09 mmol) in ACN (15 mL) was added water (0.111 mL, 6.18 mmol) and pyridin-1-ium 2,2,2-trifluoroacetate (0.717, 3.71 mmol). The resulting mixture was stirred at RT, and the reaction progress was monitored by LCMS/TLC. After the phosphoramidite was consumed, the reaction mixture containing the product was used in the next step without purification. LCMS (ES, m/z): 887.4 [M+H]⁺.

Step 2: 2-methylpropan-2-aminium (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture from Step 1 (assumed to contain 3.09 mmol of (2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyldimethylsilyloxy)-2-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-3-yl 2-cyanoethyl phosphonate) was added tert-butylamine (15.0 mL, 142 mmol) in one portion, and the resulting solution was stirred at rt for 40 min. It was concentrated, and the residue was co-evaporated with ACN (2×15 mL) to give the product, which was used for the next step without further purification. LCMS (ES, m/z): 832.3 [M−H]⁻.

Step 3. pyridin-1-ium (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of crude 2-methylpropan-2-aminium (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (˜4.2 g, ˜3.09 mmol, from step 2) in CH₂Cl₂ (37 mL) were added water (0.558 mL, 31.0 mmol) and dichloroacetic acid in CH₂Cl₂ (6%, 37 mL, 31.5 mmol). It was stirred for 40 min. Then, triethylsilane (60 mL) was added, and the solution was stirred for 1.5h. Pyridine (4.5 mL) was added to the reaction. It was concentrated. The residue was triturated with MTBE (50 ml) and Hexane (50 mL), and the supernatant was decanted. This process was repeated twice. The crude mixture was kept over P₂O₅ under reduced pressure for 20h to give a crude mixture containing the product. LCMS (ES, m/z): 532.2 [M+H]⁺.

Step 4. (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of pyridin-1-ium (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (680 mg, crude, ˜0.722 mmol) in ACN (5 mL) under Ar was added activated 4 Å molecular sieves (100 mg). The resulting mixture was stirred at rt over 30 min. (2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.805 g, 0.939 mmol) was co-evaporated with ACN (3×1 mL), re-dissolved in ACN (5 mL), and dried by adding activated 4 Å molecular sieve (100 mg). After 30 min, it was added to the previously prepared mixture containing pyridin-1-ium (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate. The mixture was stirred at rt for 1h. The reaction mixture containing the product was used in the next reaction step immediately without purification. LCMS (ES, m/z): 1288.4 [M+H]⁺.

Step 5. (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl) oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture containing the crude (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (˜1 mmol, with excess pyridinium 2,2-dichloroacetate) was added tert-butyl hydroperoxide in decane (5.5M, 0.64 mL, 3.5 mmol) dropwise. It was stirred at rt for 1h. Then, the solution was cooled to 0° C., and NaHSO₃ (250 mg) in water (5 mL) was added slowly. After 5 min, the mixture was concentrated, and the residue was purified by reverse phase (C18) chromatography eluted with 5 to 45% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 1305.6 [M+H]⁺.

Step 6. (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl) tetrahydrofuran-3-yl) oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl) oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (340 mg, 0.239 mmol) in CH₂Cl₂ (4 mL) were added water (44.5 mg, 2.468 mmol) and dichloroacetic acid (0.280 g, 2.17 mmol) in CH₂Cl (5 ml). It was stirred at rt for 30 min. Et₃SiH (4 mL) was then added, and the mixture was stirred for 1.5h. Pyridine (3 mL) was added to the reaction, and it was concentrated to give a crude product, which was used for the next step without purification. LCMS (ES, m/z): 1002.4 [M+H]⁺.

Step 7: (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl) amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-oxide

Crude (2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (1.5 g, ˜0.24 mmol) was co-evaporated with pyridine (3×5 mL) and then re-dissolved in pyridine (4 mL). To the reaction was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (160 mg, 0.865 mmol) in one portion. The resulting mixture was stirred at rt for 1h. It was used for the next reaction step directly without purification. LCMS (ES, m/z): 984.3 [M+H]⁺.

Step 8. (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2,10-dioxide

To the stirred mixture containing (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-oxide was added water (156 mg, 8.65 mmol) and iodine (81 mg, 0.321 mmol). After 10 min, the mixture was poured into a solution of NaHSO₃ (52 mg) in water (36 ml), and it was stirred for 5 min. It was cooled to 0° C., and NaHCO₃ (1.04g) was slowly added. After 5 min, EtOAc (50 mL) and Et₂O (50 ml) were added. Layers were separated, and the aq layer was extracted with EtOAc (1×30 ml). The organic layers were combined, concentrated, and purified by silica gel column chromatography eluted with 0-20% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 998.3 [M+H]⁺.

Step 9: (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino-9H-purin-9-yl}octahydro-12H-5,8-methanofuro 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide

To a stirred solution of (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2,10-dioxide (160 mg) in ACN (2 mL) was added tert-butylamine (2 mL) at rt. After 30 min, it was concentrated to give the crude product, which was used for the next step without purification. LCMS (ES, m/z): 945.2 [M+H]⁺.

Step 10: (5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide

Crude (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide (220 mg) was dissolved in a solution of MeNH₂ in EtOH (30%, 4 mL), and it was stirred at rt for 5h. Then, the volatile component was removed under reduced pressure to give a crude product, which was used for the next reaction step without purification. LCMS (ES, m/z): 773.2 [M+H]⁺ and 771.3 [M−H]⁻.

Step 11: (5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide

The crude from Step 10 was co-evaporated with pyridine (2.5 ml) and Et₃N (2.5 mL) three times. It was dissolved in pyridine (2 mL). To the solution was added Et₃N (1.51 g, 14.9 mmol) and triethylamine trihydrofluoride (1.2 g, 7.45 mmol) dropwise. The mixture was heated at 50° C. for 5h. Then, it was concentrated and purified by preparative-HPLC (T3 Prep Column, 100 Å, 5 μm, 19 mm×250 mm) eluted with 0 to 10% ACN in aq NH₄HCO₃ (50 mM) to give the product. LCMS (ES, m/z): 657.1 [M−H]⁻. ¹H-NMR: (300 MHz, DMSO-d₆+D₂O): δ 8.35 (s, 1H), 8.16 (s, 1H), 8.03 (s, 1H), 6.34 (dd, J=8.8, 5.9 Hz, 1H), 5.85 (d, J=8.3 Hz, 1H), 5.12-4.98 (m, 2H), 4.36 (d, J=3.9 Hz, 1H), 4.22 (t, J=7.2 Hz, 1H), 4.09 (s, 1H), 3.96-3.79 (m, 4H), 3.09-2.99 (m, 1H), 2.64-2.51 (m, 1H). ³¹P-NMR: (121 MHz, DMSO-d₆+D₂O): δ −1.65 (s), −2.36 (s).

Examples 2 through 19, shown in Table 1 below, were prepared according to procedures analogous to those outlined in Example 1 above using the appropriate nucleoside monomers, described as Preparations or as obtained from commercial sources.

TABLE 1 Mass Ex. Structure Name [M − H]⁻  2

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6- amino-9H-purin-9-yl)-15-fluoro-16- hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 675  3

(5R,7R,8R,12aR,14R,15aS,16R)-7,14-bis(6- amino-9H-purin-9-yl)-16-hydroxyoctahydro- 12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 657  4

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7,14-bis(6- amino-9H-purin-9-yl)-15-fluoro-16- hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 659  5

(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6- amino-9H-purin-9-yl)-16-fluoro-15- hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 675  6

(5R,7R,8R,12aR,14R,15R,15aS,18R)-7-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino- 9H-purin-9-yl)-18-hydroxyhexahydro-14H- 15,12a-(epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10(12H)-diolate 2,10-dioxide 685  7

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino- 9H-purin-9-yl)-15-fluoro-16-hydroxyoctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide 675  8

(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6- oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide 660 [M + H]⁺  9

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(4-amino- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- dihydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 672 10

(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6- oxo-1,6-dihydro-9H-purin-9-yl)-14-(4-amino- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16- hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 656 11

(5R,7R,8R,12aR,14R,15R,15aS,16S)-7-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino- 9H-purin-9-yl)-15,16-dihydroxyoctahydro-12H- 5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecine-2,10- diolate 2,10-dioxide 689 12

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino- 9H-purin-9-yl)-12a-ethynyl-15,16- dihydroxyoctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 699 [M + H]⁺ 13

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6- amino-9H-purin-9-yl)-2,10,15-trihydroxy-16- methoxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 687 14

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16R)-14-(6- amino-9H-purin-9-yl)-16-azido-2,10,15- trihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 698 15

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15-chloro-2,10,16- trihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 691 16

2-amino-9- [(5S,7R,8R,12R,12aR,14R,15R,15aS)-14-(6- amino-9H-purin-9-yl)-2,10,15-trihydroxy-12- methyl-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 671 17

2-amino-9- [(2aR,5S,6aS,7R,8R,9aR,12S,14R,14aS,15R)-8- (6-amino-9H-purin-9-yl)-5,7,12-trihydroxy-5,12- dioxidohexahydro-6aH-2a,14- (epoxymethano)furo[3,2-d]oxeto[2,3- k][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin- 15(2H,3H)-yl]-1,9-dihydro-6H-purin-6-one 685 18

2-amino-9-[(5S,7R,8R,12S,12aS,14R,15R,15aS)- 14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy- 12-methyl-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 671 19

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-15- methyl-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 655

Example 20: (5R,7R,8S,12aR,14R,15R,15aS,18R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-fluorohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10(12H-diolate 2,10-dioxide

Step 1. (2R,3514R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phenyl phosphonate

To a stirred solution of N-(9-((2R,3 S,4 S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1 g, 1.520 mmol) in pyridine (7.6 mL) under Ar was added diphenyl phosphonate (1.068 g, 4.56 mmol), and it was stirred at rt for 20 min. The reaction mixture containing the product was used for the next reaction step without purification.

Step 2. (2R,3,4R,5R)-5-((bis(4-m ethoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture from Step 1 was added water (1.5 ml) and Et₃N (1.5 mL). The mixture was stirred at rt for 20 min. Then, it was concentrated, and the residue was partitioned between CH₂Cl₂ (50 mL) and aq NaHCO₃ (5%, 20 mL). Layers were separated. The organic layer was washed with aq NaHCO₃ (5%, 2×20 mL), dried (Na₂SO₄), concentrated and purified by silica gel column chromatography using 0 to 7% MeOH in CH₂Cl₂ (1% Et₃N) to give the product. LCMS (ES, m/z): 722 [M+H]⁺. ³¹P-NMR: (162 MHz, CD₃OD): δ 2.73 (s, 1P).

Step 3. (2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of the product of Step 2 (0.9 g, 0.999 mmol) in CH₂Cl₂ (6 mL) were added water (0.180 g, 9.99 mmol) and 2,2-dichloroacetic acid (1.16 g, 8.99 mmol) in CH₂Cl₂ (10 ml). The mixture was stirred at rt for 15 min. Et₃SiH (10 mL) was added, and it was stirred for 1h. Then, pyridine (2 mL) was added, and it was concentrated. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 30% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 722 [M−H]⁻. 417.9. ¹H-NMR: (400 MHz, CD₃OD): δ 8.31 (s, 1H), 7.49 (d, J=1.6 Hz, 0.5H), 6.15 (d, J=6.8 Hz, 1H), 5.91 (d, J=1.6 Hz, 0.5H), 5.42-5.32 (m, 1.5H), 5.21 (dd, J=4.5, 1.9 Hz, 0.5H), 4.45-4.32 (m, 1H), 3.81 (d, J=3.5 Hz, 2H), 3.20 (q, J=7.4 Hz, 1H), 2.73 (p, J=6.9 Hz, 1H), 1.30 (t, J=7.3 Hz, 1.5H), 1.23 (d, J=6.9 Hz, 6H). ¹⁹F-NMR: (376 MHz, CD₃OD): δ −200.96 (s, 1F). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.41 (s, 1P).

Step 4: (2R,3S,4R,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

(2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (600 mg, 0.676 mmol) was co-evaporated with ACN (3×5 mL), re-dissolved in ACN (3 mL), dried by addition of activated 4 Å molecular sieves (150 mg) and kept under Ar. (1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl (2-cyanoethyl) diisopropylphosphoramidite (available from Exiqon (EQ-0063-1000), 235.5 mg, 0.56 mmol) and pyridinium 2,2,2-trifluoroacetate (162 mg, 0.84 mmol) were co-evaporated with ACN (3×5 mL), re-dissolved in ACN (5 mL), and dried by addition activated 4 Å molecular sieve (150 mg). After 30 min, it was added to the previously prepared mixture containing (2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate. The mixture was stirred at rt for 1h. The reaction mixture containing the product was used in the next reaction step immediately without purification. LCMS (ES, m/z): 1202.3 [M−H]⁻.

Step 5. (2R,3S,4R,5R)-5-((((((R, 3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the stirred reaction mixture from Step 4 was added tert-butyl hydroperoxide in decane (5.5M, 0.31 mL, 1.71 mmol) dropwise. The resulting mixture was stirred at rt for 1h. After 30 min, the solution was cooled to 0° C., and NaHSO₃ (150 mg) in water (5 mL) was added slowly. After 5 min, the mixture was concentrated, and the residue was purified by reverse phase (C18) chromatography eluted with 0 to 75% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 1220.1 [M+H]⁺. ¹⁹F-NMR (376 MHz, CD₃OD): δ −200.38, −202.45 (2s, 1F). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.57, 2.49 (2s, 1P); −3.52, −4.21 (2s, 1P).

Step 6. (2R,3S,4R,5R)-5-((((((S, 3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a solution of (2R,3S,4R,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (190 mg, 0.16 mmol) in CH₂Cl₂ (2.5 mL) was added water (28.8 mg, 1.6 mmol) and dichloroacetic acid in CH₂Cl₂ (0.6M, 2.5 mL). The mixture was stirred at rt for 10 min, and then Et₃SiH (4.5 mL) was added. After 1h, pyridine (0.5 mL) was added. After 10 min, the resulting mixture was concentrated to give the product, which was used for the next reaction step without purification. LCMS (ES, m/z): 917.9 [M+H]⁺. ³¹P-NMR: (162 MHz, CD₃OD): δ 2.51, 2.34 (2s, 1P); −3.46, −3.82 (2s, 1P).

Step 7: (5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10 (12H)-olate 2,10-dioxide

To pyridine (16 mL) under Ar was added diphenyl chlorophosphate (0.66 mL, 3.2 mmol). It was cooled at −40° C. and then, a solution of crude from Step 6 in CH₂Cl₂ (16 mL) was added dropwise over 20 min. The resulting mixture was stirred at −40° C. for 40 min. The reaction mixture was used in the next step without purification. LCMS (ES, m/z): 898.2 [M−H]⁻.

Step 8. (5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2,10-dioxide[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10 (12H)-diolate 2,10-dioxide

To the solution from Step 7 at 0° C. was added I₂ in pyridine/water (9/1) (3%, 1.76 mL) over 5 min. The mixture was stirred at rt for 40 min. Then, it was treated with a solution of Na₂S₂O₃.5H₂O (150 mg) in water (2 mL). After 5 min, the mixture was concentrated under reduced pressure. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 45% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 915.8 [M+H]⁺. ¹⁹F-NMR (376 MHz, CD₃OD): δ −198.70, −203.36 (2s, 1F). ³¹P-NMR (162 MHz, CD₃OD): δ −0.96, −1.75 (2s, 1P); −3.64, −4.71 (2s, 1P).

Step 9: (5R,7R,8S,12aR,14R,15R,15aS,18R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-fluorohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10(12H)-diolate 2,10-dioxide

(5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2,10-dioxide (110 mg, 0.12 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 15 mL), and the resulting solution was stirred at rt for 3h. Then, it was concentrated, and the residue was purified by preparative-HPLC (Atlantis Prep T3 Column, 19×250 mm) eluted with 0 to 9% ACN in aq NH₄HCO₃ (50 mM) to give the product. LCMS (ES, m/z): 686.9 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.14 (s, 1H), 7.85 (s, 1H), 7.80 (s, 1H), 6.01 (s, 1H), 5.99 (d, J=8.5 Hz, 1H), 5.84-5.66 (m, 1H), 5.44 (d, J=3.6 Hz, 0.5H), 5.31 (d, J=3.6 Hz, 0.5H), 4.96 (d, J=3.9 Hz, 1H), 4.84 (s, 1H), 4.65-4.53 (m, 1H), 4.33-4.15 (m, 4H), 4.10 (d, J=8.2 Hz, 1H), 3.96 (d, J=8.2 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −199.02 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ −1.89 (s, 1P), −2.49 (s, 1P).

Examples 21 through 29, as shown in Table 2 below, were prepared according to procedures analogous to those outlined in Example 20 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.

TABLE 2 Mass Ex. Structure Name [M − H] 21

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aR)-14- (6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 659 22

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-16-fluoro-2,10- dihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 659 23

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 677 24

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- oxidohexahydro-14H-15,12a-(epoxymethano)- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7(12H)-yl]- 1,9-dihydro-6H-purin-6-one 669 25

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 659 26

2-amino-9- [(2S,5R,7R,8S,10S,12aR,14R,15S,15aR,16R)-14- (6-amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 677 27

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(6- amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 641 28

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-15- methyl-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 655 29

2-amino-9-[(5S,8R,12aR,15S,15aR)-14-(6- amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy- 15-methyl-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 673

Example 30: 2-amino-7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

cGAS buffer consisted of 40 mM Tris-HCL, pH 7.5, 100 uM NaCl, 10 mM MgCl₂. cGAS enzyme was purchased from Novoprotein (Novoprotein code: SGCAS), having been expressed in E. coli and purified using a HIS tag. The calculated molecular weight was 55.3 kDa, and the sequence was:

(SEQ. ID. NO. 1) MAHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKT TPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAH REQIGGENLYFQGGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRL KCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRA YYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVI MKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAK VRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCC ENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCT QNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLID KRSKEFLTKQIEYERNNEFPVFDEF

To a vial were added Herring DNA (CAS#9007-49-2, 0.3 mg/mL in cGAS buffer; 14.8 mL) and cGAS enzyme (3.1 mg/mL in cGAS buffer; 0.78 mL), and the mixture was incubated at RT for 15 min. 7-Deaza-GTP (TriLink catalog # N-1044; 5 mM in cGAS buffer, 1.95 mL, 9.75 μmol) and ATP (5 mM in cGAS buffer, 1.95 mL, 9.75 μmol) were added, and the mixture was incubated on a Radleys Metz heater shaker set to maintain 37° C. while shaking at 250 rpm for 16h, after which the mixture was filtered and lyopholyzed. The product was purified using mass-directed reverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part#186002568] using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate. Lyopholization of the product fractions furnished the title compound. LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.37 (s, 1H), 8.10 (s, 1H), 7.56 (s, 1H), 7.24 (s, 2H), 6.94 (d, J=3.4 Hz, 1H), 6.28-6.24 (m, 3H), 6.01 (d, J=8.1 Hz, 1H), 5.86 (d, J=7.9 Hz, 1H), 5.61 (s, 1H), 5.01-4.97 (m, 1H), 4.86-4.83 (m, 1H), 4.70 (s, 1H), 4.25 (s, 1H), 4.21 (dd, J=10.4, 4.8 Hz, 1H), 4.03-3.92 (4, 3H), 3.80-3.75 (m, 1H), 3.69 (d, J=12.1 Hz, 1H), 2.76 (s, 12H), 1.02 (S, 18H).

Examples 31 to 65 in Table 3 below were made using procedures analogous to those described above for Example 30 using the appropriate nucleoside triphosphate monomers. Where necessary, the triphosphates were formed according to methods similar to those described for Preparations 23 to 26 or by submission of the requisite 5′-OH nucleoside monomer to NuBlocks LLC (Oceanside, Calif.). Example 38 was made using ATP and α-thio-GTP (BIOLOG Life Science Institute, catalog # G014/G015).

TABLE 3 Mass Ex. Structure Name [M − H]⁻ 31

2-amino-9- {(5R,7R,8R,12aR,14R,15R,15aS,16R)- 2,10,15,16-tetrahydroxy-14-[6- (methyl amino)-9H-purin-9-yl]-2,10- dioxidooctahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl}- 1,9-dihydro-6H-purin-6-one 687 32

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-chloro-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 692 33

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-amino-5-fluoro-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-2,10,15,16-tetrahydroxy- 2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 690 34

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-2-fluoro-9H-purin-9-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 691 35

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 657 36

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16R)-16- (aminomethyl)-14-(6-amino-9H-purin-9- yl)-2,10,15-trihydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 686 37

2-amino-7- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4- one 671 38

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-9H-pufin-9-yl)-2,10,15,16- tetrahydroxy-10-oxido-2-sulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 689 39

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16S)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 40

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 41

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14- (4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 672 42

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (2,6-diamino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 688 43

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 10,15,16-trihydroxy-2,10-dioxido-2- sulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 688 44

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,9-dihydro-1H-purine-2,6-dione 674 45

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 2,10,15-trihydroxy-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 656 46

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16S)-14- (6-amino-9H-purin-9-yl)-5-fluoro- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 691 47

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(2,6-diamino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 48

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 49

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14- (7-amino-1H-pyrazolo[4,3-d]pyrimidin-3- yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 50

9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6- amino-9H-purin-9-yl)-2,10,15-trihydroxy- 2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 642 51

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-amino-1H-imidazo[4,5-c]pyridin-1-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 672 52

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14- (6-amino-9H-purin-9-yl)-16-fluoro-2,10,15- trihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 675 53

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purine-6-thione 689 54

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 55

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10,15,16-tetrahydroxy- 2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 674 56

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-ethyl-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 686 57

2-amino-9- {(5R,7R,8R,12aR,14R,15R,15aS,16R)- 2,10,15,16-tetrahydroxy-14-[6-(2- methoxyethyl)-9H-purin-9-yl]-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl}- 1,9-dihydro-6H-purin-6-one 716 58

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,5-dihydro-4H-imidazo[4,5-c]pyridin-4- one 657 59

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (2-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 60

4-amino-7- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 697 61

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,5-dihydro-4H-imidazo[4,5-c]pyridin-4- one 656 62

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS)-14-(6- amino-9H-purin-9-yl)-2,10,15-trihydroxy- 16-methylidene-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]/ pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 669 63

7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,7-dihydro-4H-imidazo[4,5- d][1,2,3]triazin-4-one 659 64

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (7-amino-3H-imidazo[4,5-b]pyridin-3-yl)- 2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 672 65

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)- 14-(6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,5-dihydro-4H-imidazo[4,5-d]pyridazin-4- one 658

Example 66: 1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

cGAS buffer consisted of 40 mM Tris-HCL, pH 7.5, 100 uM NaCl, 10 mM MgCl₂. cGAS enzyme was purchased from Novoprotein (Novoprotein code: SGCAS), having been expressed in E. coli and purified using a HIS tag. The calculated molecular weight was 55.3 kDa, and the sequence was:

(SEQ. ID. NO. 1) MAHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKT TPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAH REQIGGENLYFQGGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRL KCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRA YYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVI MKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAK VRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCC ENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCT QNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLID KRSKEFLTKQIEYERNNEFPVFDEF

To a vial were added Herring DNA (CAS#9007-49-2, 0.3 mg/mL in cGAS buffer; 15.2 mL) and cGAS enzyme (3.1 mg/mL in cGAS buffer; 0.8 mL), and the mixture was incubated at RT for 15 min. ATP (5 mM in cGAS buffer, 2.0 mL, 10 μmol), 7-deaza-8-aza-ITP (5 mM in cGAS buffer, 2.0 mL, 10 μmol), and DMSO (5 mL) were added, and the mixture was incubated on a Radleys Metz heater shaker set to maintain 37° C. while shaking at 250 rpm for 3d. The mixture was filtered, lyopholyzed, and purified by reverse phase HPLC (eluting acetonitrile/water gradient with 100 mM TEAA modifier, linear gradient) to afford the title compound as the TEA salt. LCMS (ES, m/z): 658 [M−H]⁻. ¹H NMR (600 MHz, D₂O): δ 8.36 (s, 1H), 8.34 (s, 1H), 8.13 (s, 1H), 7.51 (s, 1H), 6.41 (d, J=8.2 Hz, 1H), 6.24 (s, 1H), 5.69 (m, 1H), 5.42 (m, 1H), 4.88 (d, J=4.4 Hz, 1H), 4.66 (d, J=4.1 Hz, 1H), 4.51 (m, 1H), 4.43 (m, 2H), 4.23 (m, 2H), 4.01 (m, 1H).

Examples 67 to 74 in Table 4 below were made using procedures analogous to those described above for Example 66 using the appropriate nucleoside triphosphate monomers.

Where necessary, the triphosphates were formed according to methods similar to those described for Preparations 23 to 26 or by submission of the requisite 5′-OH nucleoside monomer to NuBlocks LLC (Oceanside, Calif.).

TABLE 4 Mass Ex Structure Name [M − H]⁻ 67

9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 2,10,15-trihydroxy-2,10-dioxidooctahydro- 12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 641 68

7-[(5R,7S,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7- yl]imidazo[2,1-f][1,2,4]triazin-4(3H)-one 658 69

5-amino-3- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 674 70

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)- 2,10,15,16-tetrahydroxy-14-(6-methyl-9H- purin-9-yl)-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 672 71

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 691 72

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)- 2,10,15,16-tetrahydroxy-2,10-dioxido-14- (9H-purin-9-yl)octahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 658 73

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14- (4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin- 1-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 673 74

3-[(5R,7S,8R,12aR,14R,15R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,15,16- tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7- one 658

Example 75: 2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][11,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1: 2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To a stirred solution of 2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-azido-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Example 14, 4.0 mg, 0.0055 mmol) in absolute EtOH (1.0 mL) and deionized water (1.0 mL) was added palladium on carbon (1.0 mg, 10 wt. % loading) in one portion under Ar at RT. The reaction vessel was then flushed with hydrogen gas and attached to a hydrogen gas balloon. The reaction mixture was left to stir for 48h, filtered, and concentrated to afford the title compound. LCMS (ES, m/z): 672 [M−H]⁻. (600 MHz, DMSO-d₆) δ 10.70 (s, 1H), 8.42 (s, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.70 (s, 1H), 7.29 (br, 2H), 6.56 (br, 2H), 6.00 (d, J=8.3 Hz, 1H), 5.89 (d, J=8.5 Hz, 1H), 5.21 (s, 1H), 5.04 (t, J=6.0 Hz, 1H), 4.16 (s, 1H), 4.05 (dd, J=10.5, 5.0 Hz, 1H), 4.00 (s, 1H), 3.77 (d, J=4.1 Hz, 1H), 3.67 (m, 2H). ³¹P NMR: (202 MHz, DMSO-d₆): δ −0.4 (s), 2.0 (s).

Alternatively, Example 75 may be prepared from the requisite monomers, according to a method similar to that described above for Example 30.

Example 76: 9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carboxamide

To a stirred solution of 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-chloro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one, (Example 32, 16 mg, 0.018 mmol) in DMSO (1.7 mL) was added sodium cyanide (8.0 mg, 0.16 mmol) in one portion under Ar at RT. The reaction mixture was heated to 80° C. and left to stir at the same temperature for 3h, cooled to ambient temperature, then quenched with cold acetic acid (15 uL). The mixture was filtered and lyophilyzed. The product was purified using mass-directed reverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part#186002568] using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate. Lyophilization of the product fractions furnished 9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carbonitrile. LCMS (ES, m/z): 683 [M−H]⁻.

To a stirred suspension of 9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carbonitrile (3.0 mg, 0.003 mmol) in deionized water (338 uL) was added Hydrido(dimethylphosphinous acid-kP)[hydrogen bis(dimethylphosphinito-kP)]platinum(II) (1.0 mg, 0.002 mmol). The reaction mixture was heated to 85° C. and left to stir at the same temperature for 6h, cooled to ambient temperature, filtered and lyopholyzed. The product was purified using mass-directed reverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part#186002568] using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate. Lyopholization of the product fractions furnished 9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carboxamide. LCMS (ES, m/z): 701 [M−H]⁻. ¹H NMR (500 MHz, DMSO): δ 10.58 (s, 1H), 9.06 (s, 1H), 8.36 (s, 1H), 8.05 (s, 1H), 7.91 (s, 1H), 7.74 (s, 1H), 6.55 (br, 4H), 6.07 (d, J=7.7 Hz, 1H), 5.81 (d, J=6.0 Hz, 1H), 5.77 (m, 1H), 5.04 (m, 1H), 4.96 (d, J=4.5 Hz, 1H), 4.60 (m, 1H), 4.27 (m, 1H), 4.07-4.04 (m, 2H), 3.99-3.75 (m, 2H). ³¹P NMR: (202 MHz, DMSO): δ 1.9 (s), −0.8 (s).

Examples 77, 78, 79, 80: 2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1-4)

Step 1. (2R,3R)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a flask was added (2R,3R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (4.00 g, 4.76 mmol), MeCN (23.65 ml), and water (0.158 ml). Pyridine trifluoroacetate (1.104 g, 5.71 mmol) was added, and the reaction was stirred at rt for 1h. Tert-butylamine (20.02 ml, 190 mmol) was then added, and stirring was continued at rt for 1h, after which time the reaction was partitioned between hexanes and acetonitrile. The acetonitrile layer was collected and concentrated under vacuum. DCM (39.9 ml) and water (0.798 ml) were added, followed by dichloroacetic acid (55.1 ml, 33.3 mmol), and the solution was stirred for 20 min at rt, after which time triethylsilane (133 ml, 833 mmol) was added, and the reaction was stirred for a further 2h at rt. The reaction was cooled to 0° C., and pyridine (5.39 mL 66.6 mmol) was added. Then the mixture was concentrated under reduced pressure to give the title compound, which was not purified further. LCMS (ES, m/z): 400 [M−H]⁻.

Step 2: O-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl) 0-(((2S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogen phosphorothioate

To a flask was added (2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (335 mg, 0.836 mmol) and MeCN (20 mL), and then the solution was concentrated under reduced pressure. This process was repeated 2×, and then MeCN (8 mL) was added, followed by activated 4 Å sieves. The mixture was stirred for 20 min at rt. (2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl) oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (825 mg, 0.836 mmol) was dissolved in MeCN (5 mL). Molecular sieves (4 Å) were added, and the mixture was stirred for 30 min at rt, after which time this solution was transferred to the hydrogen phosphonate solution and 2×1.5 mL portions of MeCN were used to complete the transfer. After stirring 30 min at rt, ((dimethylamino-methylidine)amino)-3H-1,2,4-dithiazoline-3-thione (189 mg, 0.919 mmol) was added. After stirring 5 min at rt, the mixture was concentrated under reduced pressure and purified using reverse phase HPLC with a 10-100% gradient of MeCN and 100 mM aqueous triethylammonium acetate. The product-containing fractions were collected and lyophilized, during which time the cyanoethyl protecting group was cleaved, to furnish O-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl) O-(((2S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogen phosphorothioate. LCMS (ES, m/z): 1264 [M−H]⁻.

Step 3: N-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide (Diastereomers 14)

To a flask containing O-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-3-yl)O-(((2 S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogen phosphorothioate (581 mg, 0.440 mmol) was added DCM (8.81 mL), water (0.079 mL, 4.40 mmol) and then dichloroacetic acid (8.74 mL, 5.28 mmol). The mixture was stirred for 15 min at rt, and then triethylsilane (10.97 mL, 68.7 mmol) was added. The mixture was stirred at rt for 1.5h and then concentrated under reduced pressure. The mixture was dissolved in pyridine (10 mL) and then concentrated under reduced pressure. This process was repeated 2×, and then the resulting sample was dissolved in pyridine (17 ml) and 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinan-2-one (81 mg, 0.440 mmol) was added in 1 portion at rt. After stirring for 30 min at rt, additional 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinan-2-one (81 mg, 0.440 mmol) was added. This sequence was repeated twice, and then water (238 μl, 13.19 mmol) and 3H-1,2-benzodithiol-3-one (111 mg, 0.660 mmol) were added. The mixture was stirred for 1h at rt and then partitioned between water (10 mL) and 1:1 EtOAc/ether (10 mL). The layers were separated, and the aqueous phase was extracted with 1:1 EtOAc/ether (3×10 mL). The combined organic phases were dried (MgSO₄), filtered, and concentrated under reduced pressure. Reverse phase HPLC purification (gradient of 30-100% MeCN and 100 mM aqueous triethylammonium acetate) furnished 4 diastereomers of N-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl) silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide, all of which showed LCMS (ES, m/z): 976 [M−H]⁻.

Step 4: 2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1-4)

To a flask containing N-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide (fastest eluting peak, 7.4 mg, 7.57 tmol) was added methylamine (33% in Et0H) (1 mL, 8.03 mmol), and the mixture was stirred at rt for 4h, after which time the mixture was concentrated under reduced pressure. Pyridine (1 mL) was added, and the mixture was concentrated under reduced pressure. Then, pyridine (0.5 ml), triethylamine (0.104 ml, 0.746 mmol) and triethylamine trihydrofluoride (0.030 ml, 0.187 mmol) were added, and the mixture was stirred at 50° C. for 16h, after which time the mixture was cooled to rt and concentrated under reduced pressure. Purification by reverse phase HPLC (gradient of acetonitrile and 100 mM aqueous triethylammonium acetate) furnished Example 77, 2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, Deuterium Oxide) δ 8.06 (s, 1H), 8.03 (s, 1H), 7.41 (d, J=3.8 Hz, 1H), 6.25 (d, J=3.7 Hz, 1H), 6.17 (d, J=2.6 Hz, 1H), 5.68 (d, J=7.5 Hz, 1H), 5.42-5.36 (m, 2H), 5.10-5.06 (m, 1H), 4.83-4.81 (m, 1H), 4.51-4.48 (m, 1H), 4.36-4.33 (m, 1H), 4.28 (dt, J=10.1, 4.9 Hz, 1H), 4.06-3.94 (m, 2H), 3.03 (q, J=7.3 Hz, 12H), 2.44-2.40 (m, 2H), 1.11 (t, J=7.3 Hz, 18H).

The other diastereomers from Step 3 were individually processed in an analogous manner to afford three additional diastereomeric products:

Example 78: 2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, Deuterium Oxide) δ 8.04 (s, 1H), 7.75 (s, 1H), 7.50 (d, J=3.6 Hz, 1H), 6.19-6.17 (m, 1H), 6.16 (d, J=3.7 Hz, 1H), 5.65 (d, J=7.4 Hz, 1H), 5.63-5.57 (m, 1H), 5.14 (td, J=7.8, 4.4 Hz, 1H), 4.54 (d, J=4.2 Hz, 1H), 4.52-4.46 (m, 1H), 4.33 (d, J=8.9 Hz, 1H), 4.27 (dd, J=11.9, 3.1 Hz, 1H), 4.18-4.15 (m, 1H), 3.96 (dd, J=11.4, 3.6 Hz, 2H), 3.04 (q, J=7.3 Hz, 12H), 2.44-2.36 (m, 2H), 1.11 (t, J=7.3 Hz, 18H).

Example 79: 2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, Deuterium Oxide) δ 8.00 (s, 1H), 7.97 (s, 1H), 7.19 (d, J=3.8 Hz, 1H), 6.15 (d, J=3.8 Hz, 2H), 5.66 (d, J=7.3 Hz, 1H), 5.32 (dq, J=9.9, 7.2 Hz, 1H), 4.95 (td, J=8.6, 4.6 Hz, 1H), 4.81 (d, J=4.4 Hz, 1H), 4.50 (d, J=8.0 Hz, 1H), 4.32 (d, J=7.8 Hz, 1H), 4.25 (dt, J=12.0, 3.6 Hz, 1H), 4.05-3.97 (m, 3H), 3.03 (q, J=7.3 Hz, 12H), 2.51-2.41 (m, 2H), 1.11 (t, J=7.3 Hz, 18H).

Example 80: 2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, Deuterium Oxide) δ 8.00 (s, 1H), 7.72 (s, 1H), 7.23 (d, J=3.8 Hz, 1H), 6.14 (s, 1H), 6.12 (d, J=3.7 Hz, 1H), 5.64 (d, J=6.9 Hz, 1H), 5.47 (dq, J=14.2, 7.0 Hz, 1H), 5.05 (td, J=8.0, 4.5 Hz, 1H), 4.52 (d, J=4.4 Hz, 1H), 4.49 (dt, J=7.4, 2.7 Hz, 1H), 4.33-4.27 (m, 2H), 4.19 (ddd, J=11.5, 8.5, 3.0 Hz, 1H), 4.00 (dd, J=11.5, 3.9 Hz, 1H), 3.94 (ddd, J=11.6, 5.6, 2.2 Hz, 1H), 3.01 (q, J=7.3 Hz, 12H), 2.49-2.40 (m, 2H), 1.10 (t, J=7.3 Hz, 18H).

Example 81: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1)

Step 1: (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (3 g, 3.09 mmol) in MeCN (15 mL) at 25° C. was added H₂O (0.111 mL, 6.18 mmol) and pyridin-1-ium 2,2,2-trifluoroacetate (0.717 g, 3.71 mmol). The resulting mixture was stirred at 25° C. for 20 min. The reaction progress was monitored by LCMS/TLC. After the phosphoramidite starting material was consumed, the reaction mixture that containing the desired product (major) was used for the next step without any after-treatment. LCMS (ES, m/z): 887.4 [M+H]⁺.

Step 2: (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of the product of Step 1 (15.0 mL, 142 mmol) from the previous reaction was added tert-butylamine in one portion, and it was stirred at 25° C. for 40 min. The resulting solution was concentrated in vacuo. The residue was co-evaporated with dry MeCN (two times, 15 mL each), used for the next step without purification. LCMS (ES, m/z): 832.3 [M−H]⁻.

Step 3: (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of the product of Step 2 in CH₂Cl₂ (37 mL) was added H₂O (0.558 mL, 31.0 mmol) and 6% DCA in CH₂Cl₂ (37 mL, 31.5 mmol) dropwise. The resulting mixture was stirred at 25° C. for 40 min, then Et₃SiH (60 mL) was added, and the reaction mixture was stirred for 1.5h. Pyridine (4.5 mL, 2eq to DCA) was added to the reaction. The resulting solution was stirred at 25° C. for 5 min and then concentrated in vacuo. The residue was triturated with MTBE/hexane (100 mL, v/v, 1/1), and the supernatant was decanted. This process was repeated two more times. The final residue was concentrated at reduced pressure and was used for the next step without purification. LCMS (ES, m/z): 532.18 [M+H]⁺.

Step 4. (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of the product of Step 3 (0.704 g, 0.722 mmol) in MeCN (5 mL) under Ar was added activated 4 Å molecular sieve (200 mg), and the mixture was stirred at RT over 30 min. (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropyl-phosphoramidite (0.822 g, 0.939 mmol) was twice co-evaporated with dry MeCN (3 mL). Activated 4 Å molecular sieve (200 mg) was added. After 30 min, the phosphoramidite solution was transferred into the solution of the product of Step 3 by syringe. The resulting mixture was stirred at RT for 20 min. The desired product was detected by TLC/LCMS, and the reaction solution was used for the next reaction without purification. LCMS (ES, m/z): 1306.7 [M+H]⁺.

Step 5. (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture containing the product of Step 4 (˜0.722 mmol) under Ar was added (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (163 mg, 0.794 mmol) in one portion, and the mixture was stirred at RT for 30 min. The reaction progress was monitored by TLC/LCMS. After the consumption of the starting phosphite, the reaction mixture was concentrated in vacuo, and the residue was purified by reverse phase chromatography (X-Bridge BEH130 Prep C18) eluting with 5 to 95% MeCN in H₂O (0.04% NH₄HCO₃). The product-containing fractions were combined and concentrated under reduced pressure to 2/3 volume. NaCl (10g) was added, and the aqueous mixture was extracted with EtOAc/Et₂O (v/v, 1/1, 3×80 mL). The combined organic layers were dried (Na₂SO₄) and concentrated. LCMS (ES, m/z): 1339.5 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 67.83 (d, J=43.9 Hz), 2.81 (d, J=15.7 Hz).

Step 6. (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of the product of Step 5 (180 mg, 0.128 mmol) in CH₂Cl₂ (7 mL) was added 2,2-dichloroacetic acid in CH₂Cl₂ (2.47 mg, 1.15 mmol) and H₂O (22.97 mg, 1.28 mmol). After stirring at RT for 20 min, Et₃SiH (4.5 mL) was added. After 2h, pyridine (1 mL) was added, and the mixture was stirred for 10 min. After removal of volatiles, the product was used for the next reaction step without purification. LCMS (ES, m/z): 1036.4 [M+H]⁺.

Step 7: (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-sulfide

The product of Step 6 (570 mg) was co-evaporated with dry pyridine (1 mL each, three times). To the mixture in dry pyridine (4 mL) at RT under Ar was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (71 mg, 0.384 mmol) in one portion. The resulting mixture was stirred for 40 min. The reaction progress was monitored by TLC/LCMS. The desired product as a mixture of diastereomers was observed, and the product was used for the next reaction step directly. LCMS (ES, m/z): 1018.5 [M+H]⁺.

Step 8: Diastereomeric mixtures (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-thiolate 10-oxide 2-sulfide (A1) and (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-thiolate 10-oxide 2-sulfide (A2)

To the stirred mixture of the product of Step 7 was added H₂O (69.2 mg, 3.84 mmol) and 3H-benzo[c][1,2]dithiol-3-one (32.3 mg, 0.192 mmol). The mixture was stirred at RT for 40 min. The reaction progress was monitored by TLC/LCMS. After the reaction completed, the reaction mixture was poured into aq NaHCO₃ (0.14g NaHCO₃ in 5 mL H₂O) and stirred for 5 min. The resulting mixture was extracted with EtOAc/ether (v/v, 1/1, 3×15 mL). The combined organic layers were dried (Na₂SO₄), and purified by silica gel chromatography eluted with 0 to 15% MeOH in CH₂Cl₂ to give products: mixture of diastereomers A1 (eluted out at 5.5% MeOH in CH₂Cl₂); mixture of diastereomers A2 (eluted out at 9.8% MeOH in CH₂Cl₂); (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl) oxy)methyl)-4-((tert-butyldimethyl silyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (eluted out at 12.6% MeOH in CH₂Cl₂). Mixture A1: LCMS (ES, m/z): 1050.30 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 66.34 (s), 64.63 (s). Mixture A2: LCMS (ES, m/z): 1050.30 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 65.94, 64.17, 62.55, 61.28.

Step 9: Diastereomers (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (BI) and (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (B2)

To a stirred suspension of crude A1 (95 mg, ˜0.09 mmol) from the previous step in MeCN (1 mL) at RT under Ar was added tert-butylamine (1.5 mL). After 30 min, volatile components were removed in vacuo. The residue was purified by reverse phase prep-HPLC (X-Bridge BEH130 Prep C18) eluted with 25 to 45% MeCN in aq NH₄HCO₃ (10 mM) over 8 min to give compound B2 as a single diastereomer (T_(R)=5.97 min). LCMS (ES, m/z): 891.4 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ 8.44 (s, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 6.37 (d, J=14.0 Hz, 1H), 5.95 (d, J=8.0 Hz, 1H), 5.65-5.54 (m, 1H), 5.26-5.10 (m, 2H), 4.57-4.41 (m, 4H), 4.24 (s, 1H), 4.01 (d, J=11.5 Hz, 1H), 3.89 (d, J=11.8 Hz, 1H), 2.75-2.63 (m, 1H), 1.04-0.91 (m, 15H), 0.28-0.24 (m, 6H). ³¹P-NMR (121 MHz, CD₃OD): δ 57.10 (s), 53.1 (s).

Step 10: (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-6-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2, O-bis(thiolate) 2,10-dioxide

To a stirred solution of compound B2 (13 mg, 0.013 mmol) from the previous step was added a solution of MeNH₂ in EtOH (0.6 mL, 30% by weight). The mixture was stirred at RT for 12h. The volatile components were removed under reduced pressure, and the residue containing product compound was used for the next reaction step without purification. LCMS (ES, m/z): 823.15 [M+H]⁺.

Step 11: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1)

The crude product from Step 10 (25 mg) was co-evaporated with pyridine/Et₃N (v/v, 3/1, 1 mL each, three times) and then dissolved in pyridine (0.15 mL). The mixture was charged with Ar and Et₃N (0.20 mL) and triethylamine trihydrofluoride (56.4 mg, 0.350 mmol) were added. The resulting solution was warmed at 50° C. for 6h. The reaction progress was monitored by TLC/LCMS. After completion of the reaction, the mixture was concentrated in vacuo and then, co-evaporated with MeCN (three times, 1 mL each). The residue was purified by reverse phase prep-HPLC (X-Bridge BEH130 Prep C18) eluted with 0 to 22% MeCN in aq NH₄HCO₃ (50 mM) over 15 min to give the product compound (T_(R)=8.3 min). LCMS (ES, m/z): 708.95 [M+H]⁺. ¹H-NMR (400 MHz, D₂O): δ 8.18 (s, 1H), 8.16 (s, 1H), 7.77 (s, 1H), 6.37 (d, J=14.3 Hz, 1H), 5.86 (d, J=8.4 Hz, 1H), 5.61-5.54 (m, 1.5H), 5.43 (s, 0.5H), 5.27-5.12 (m, 2H), 4.59 (d, J=3.6 Hz, 1H), 4.47 (t, J=12.9 Hz, 2H), 4.36 (d, J=4.8 Hz, 1H), 4.04 (dd, J=23.2, 12.0 Hz, 2H). ³¹P-NMR (162 MHz, D₂O): δ 55.63 (s), 51.55 (s).

Example 82: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2)

Step 1: Diastereomers (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (B3) and (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (B4)

To a stirred suspension of crude A2 (105 mg, ˜0.1 mmol) from Example 81, Step 8 in MeCN (1 mL) under Ar was added tert-butylamine (1.5 mL), and the mixture was stirred at RT for 30 min. The volatile components were removed in vacuo. The residue was purified by reverse phase prep-HPLC (X-Bridge BEH130 Prep C18) eluted with 25 to 40% MeCN in aq NH₄HCO₃ (10 mM) over 10 min to give two diastereomeric compounds, B3 (T_(R)=6.12 min, 0.025 mmol) and B4 (T_(R)=7.45 min, 0.021 mmol).

Compound B3: LCMS (ES, m/z): 995.3 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ 8.82 (s, 1H), 8.72 (s, 1H), 8.65 (s, 1H), 8.20-8.13 (m, 2H), 7.66-7.54 (m, 3H), 6.47 (d, J=14.0 Hz, 1H), 6.09 (d, J=8.4 Hz, 1H), 5.96-5.95 (m, 0.5H), 5.81-5.78 (m, 0.5H), 5.52-5.36 (m, 2H), 4.64-4.56 (m, 2H), 4.48-4.43 (m, 1H), 4.37-4.30 (m, 1H), 4.25-4.22 (m, 1H), 4.17-4.10 (m, 1H), 3.98 (d, J=11.7 Hz, 1H), 2.65 (p, J=6.8 Hz, 1H), 1.12 (d, J=6.8 Hz, 3H), 0.98-0.95 (m, 12H), 0.22 (d, J=8.0 Hz, 6H). ³¹P-NMR (121 MHz, CD₃OD): δ 56.96 (s), 55.90 (s).

Compound B4: LCMS (ES, m/z): 995.4 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ 8.97 (s, 1H), 8.68 (s, 1H), 8.24-8.22 (m, 3H), 7.59 (ddd, J=14.5, 7.9, 6.2 Hz, 3H), 6.46 (d, J=13.0 Hz, 1H), 5.99 (d, J=8.3 Hz, 1H), 5.67-5.57 (m, 1H), 5.45-5.33 (m, 2H), 4.56 (dd, J=13.5, 4.9 Hz, 2H), 4.47-4.38 (m, 2H), 4.25 (t, J=3.5 Hz, 1H), 4.07 (d, J=11.3 Hz, 1H), 3.94 (d, J=11.0 Hz, 1H), 2.75 (p, J=6.8 Hz, 1H), 1.04 (d, J=6.8 Hz, 3H), 0.95 (s, 12H), 0.23 (d, J=5.2 Hz, 6H). ³¹P-NMR (121 MHz, CD₃OD): δ 56.81 (s), 54.76 (s).

Step 2: (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2, O-bis(thiolate) 2,10-dioxide

Compound B3 from Step 1 (25 mg, 0.025 mmol) was dissolved in a solution of MeNH₂ in EtOH (1 mL, 30% by weight), and the mixture was stirred at RT for 12h. The reaction progress was monitored by TLC/LCMS. After the reaction was complete, the volatile components were removed in vacuo, and the residue containing the crude product was used for the next reaction step without purification. LCMS (ES, m/z): 823.25 [M+H]⁺.

Step 3: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2)

The crude product of Step 2 (35 mg) was co-evaporated with pyridine/Et₃N (v/v, 3/1, 1 mL each, three times) and then re-dissolved in pyridine (0.4 mL). The mixture was charged with Ar and Et₃N (0.34 mL, 2.4 mmol), and triethylamine trihydrofluoride (97 mg, 0.6 mmol) were added.

The resulting solution was warmed at 50° C. for 6h. Then, the mixture was concentrated at reduced pressure and then co-evaporated with MeCN (3×1 mL). The residue was purified by reverse phase prep-HPLC (X-Bridge BEH130 Prep C18) eluted with 0 to 10% MeCN in aq NH₄HCO₃ (50 mM) over 14 min to give the product compound (T_(R)=9.2 min). LCMS (ES, m/z): 709.00 [M+H]⁺. H-NMR (400 MHz, DMF-d₇+D₂O): δ 8.68 (s, 1H), 8.61 (s, 1H), 8.45 (s, 1H), 6.57 (d, J=14.8 Hz, 1H), 6.27-6.25 (m, 1.5H), 6.15-6.13 (m, 0.5H), 5.72-5.68 (m, 1H), 5.56-5.54 (m, 1H), 4.85-4.83 (m, 1H), 4.71-4.69 (m, 1H), 4.52-4.43 (m, 4H), 4.27-4.24 (m, 1H). ³¹P-NMR (162 MHz, DMF-d₇+D₂O): δ 56.03 (s), 53.37 (s). ¹⁹F-NMR (376 MHz, DMF-d₇+D₂O): δ −205.44 (s).

Example 83: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3)

Step 1: (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide

Compound B4 ((5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide, 21 mg, 0.021 mmol) from Example 82, Step 1 was dissolved in a solution of MeNH₂ in EtOH (1 mL, 30% by weight), and the mixture was stirred at RT for 12h. The reaction progress was monitored by TLC/LCMS. After the reaction was complete, the volatile components were removed in vacuo, and the product was used for the next reaction step without purification. LCMS (ES, m/z): 823.25 [M+H]⁺.

Step 2: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3)

The crude product of Step 1 (31 mg) was co-evaporated with pyridine/Et₃N (v/v, 3/1, 3×1 mL) and then, re-dissolved in pyridine (0.4 mL). The mixture was charged with Ar and Et₃N (0.28 mL, 2.0 mmol) and triethylamine trihydrofluoride (81 mg, 0.5 mmol) were added. The resulting solution was warmed at 50° C. for 6h. The mixture was concentrated at reduced pressure and then co-evaporated with MeCN (3×1 mL). The residue was purified by reverse phase prep-HPLC (X-Bridge BEH130 Prep C18) eluting with 0 to 10% MeCN in aq NH₄HCO₃ (50 mM) over 14 min to give the product compound (T_(R)=10.1 min). LCMS (ES, m/z): 709.00 [M+H]⁺. H-NMR (400 MHz, DMF-d₇+D₂O): δ 8.73 (s, 1H), 8.28-8.20 (m, 2H), 6.55 (d, J=14.8 Hz, 1H), 6.25-5.85 (m, 3H), 5.62-5.56 (m, 1H), 4.76 (s, 1H), 4.62-4.60 (m, 2H), 4.49-4.41 (m, 3H), 4.18-4.15 (m, 1H). ³¹P-NMR (162 MHz, DMF-d₇+D₂O): δ 56.09 (s), 54.75 (s). ¹⁹F-NMR (376 MHz, DMF-d₇+D₂O): δ −203.33 (s).

Examples 84 through 116, shown in Table 5 below, were prepared according to procedures analogous to those outlined in Examples 77 through 83 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.

TABLE 5 Mass Ex. Structure Name [M − H]⁻  84

2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,16-trihydroxy- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 691 [M + H]⁺  85

2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-14- (6-amino-9H-purin-9-yl)-2,10,16-trihydroxy- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 691 [M + H]⁺  86

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9- yl)-2,10,15-trihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 689  87

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10,15-trihydroxy- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 689  88

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10,15-trihydroxy- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 689  89

2-amino-9-[(2R,5R,7R,8R,10,R, 12aR,14R,15S,15aR,16R)-14-(6-amino-9H- purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 707  90

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15-fluoro-2,10,16- trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 707  91

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15-fluoro-2,10,16- trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 707  92

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15-fluoro-2,10,16- trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 4) 707  93

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7(12H)-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 717  94

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 717  95

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 719 [M + H]⁺  96

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15-fluoro-16-hydroxy- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 723  97

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16S)-14-(6-amino-9H- purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxido- 2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 723  98

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15-fluoro-16-hydroxy- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 723  99

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15-fluoro-16-hydroxy- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 4) 723 100

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 733 101

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclo- tetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 733 102

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 3) 733 103

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6- amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro- 2,10,15-trihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 731 104

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6- amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro- 2,10,15-trihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 731 105

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6- amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro- 2,10,15-trihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 731 106

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16R)-14-(6- amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro- 2,10,15-trihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 731 107

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16R)-14-(6- amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro- 2,10,15-trihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 731 108

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 706 109

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 2) 706 110

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 3) 706 111

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 706 112

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 2) 706 113

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 3) 706 114

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- hydroxy-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 4) 706 115

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15-fluoro-16-hydroxy-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 725 116

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15-fluoro-16-hydroxy-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 725

Examples 117 and 118: 2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) and 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2)

Step 1: 2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl) oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

((2R,3 S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (1058 mg, 1.234 mmol) was co-evaporated with dry ACN (3×5 mL), re-dissolved in ACN (10 mL) under Ar, and dried by adding activated 4 Å molecular sieve (200 mg). (2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (430 mg, 1.03 mmol) and pyridinium 2,2,2-trifluoroacetate (298 mg, 1.54 mmol) were co-evaporated with ACN (3×5 mL) and then re-dissolved in ACN (10 mL), and dried by adding activated 4 Å molecular sieve (200 mg). After 30 min, it was added to the previously prepared mixture containing ((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite. It was stirred at rt for 30 min. The reaction mixture was used for the next reaction step without purification. LCMS (ES, m/z): 1173.8 [M−H]⁻.

Step 2. (2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture from Step 1 was added (E)-N,N-dimethyl-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (DDTT, 0.232 g, 1.13 mmol) in one portion. The mixture was stirred at rt for 1h. It was concentrated to give a crude sample containing the product, which was used for the next reaction step without purification. LCMS (ES, m/z): 1205.8 [M−H]⁻.

Step 3. (2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a solution of the crude from Step 2 in CH₂Cl₂ (15 mL) was added water (0.2 mL, 10 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6M, 15 mL, 9 mmol). After 30 min, triethylsilane (28 mL) was added, and it was stirred for 1.5h. Then, pyridine (1.4 mL) was added. It was concentrated, and the residue was purified by reverse phase (C18) chromatography eluted with 0 to 43% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 905.8 [M+H]⁺. ¹H-NMR (300 MHz, CD₃OD): δ 8.71-8.44 (m, 2H), 8.21-8.03 (m, 3H), 7.80 (d, J=10.4 Hz, 0.5H), 7.66-7.61 (m, 1H), 7.54 (t, J=7.6 Hz, 2H), 6.61-6.42 (m, 1H), 6.14 (dd, J=13.2, 6.0 Hz, 1H), 5.68 (d, J=9.9 Hz, 0.5H), 5.60-5.19 (m, 3H), 4.69-4.36 (m, 3H), 4.36-4.17 (m, 3H), 3.92-3.64 (m, 2H), 3.13-2.55 (m, 5H), 1.19 (dd, J=6.9, 2.1 Hz, 6H). ¹⁹F-NMR (282 MHz, CD₃OD): δ −202.55, −202.75 (d, 1F). ³¹P-NMR (121 MHz, CD₃OD): δ 66.91, 66.69 (2s, 1P); 2.66, 2.60 (2s, 1P).

Step 4: (5R,7R,8S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-sulfide

Diphenyl phosphorochloridate (2375 mg, 8.84 mmol) was added to pyridine (45 ml) at −30° C. To the solution at −30° C. was added (2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl) oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (400 mg, 0.44 mmol) in CH₂Cl₂ (45 mL) dropwise over 20 min. The resulting mixture was stirred at −30° C. for 40 min. The reaction mixture was used for the next reaction step immediately without purification. LCMS (ES, m/z): 887.8 [M+H]⁺.

Step 5: (5R,7R,8S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2,10-disulfide

To the mixture from Step 4 at −30° C. was added 3H-benzo[c][1,2]dithiol-3-one (112 mg, 0.663 mmol) and water (279 μL, 15.5 mmol). After stirring at rt for 1h, the mixture was poured into a solution of Na₂S₂O₃.5H₂O (280 mg) in water (10 mL) at 0° C. It was stirred at rt for 5 min, and the mixture was concentrated under reduced pressure. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 919.8 [M+H]⁺. ¹⁹F-NMR (376 MHz, CD₃OD): δ −198.51, −198.98, −200.16 (3s, 1F). ]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 65.90, 65.09, 63.64, 62.95, 57.26, 56.50 (6s, 2P).

Step 6. Diastereomers (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl) amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-A), (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-B), and (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-C)

To a solution of (5R,7R,8 S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2,10-disulfide (265 mg, 0.288 mmol) in ACN (5 mL) at rt was added tert-butylamine (5 mL, 0.29 mmol). The reaction mixture was stirred for 10 min. Then, volatile components were removed under reduced pressure. The residue was purified by preparative-HPLC (T3 Prep Column, 19 mm×250 mm) eluted with 5 to 20% ACN in aq NH₄HCO₃ (50 mM) over 21 min.

The first fractions (T_(R): 8.95 min) gave (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-A). LCMS (ES, m/z): 866.7 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.74 (s, 1H), 8.71 (s, 1H), 8.26 (s, 1H), 8.07 (d, J=7.5 Hz, 1H), 7.65 (d, J=7.7 Hz, 1H), 7.57 (t, J=7.5 Hz, 2H), 6.60-6.34 (m, 1H), 5.94 (d, J=8.6 Hz, 1H), 5.91-5.66 (m, 1H), 5.46-5.16 (m, 2H), 4.50 (d, J=27.0 Hz, 1H), 4.27 (d, J=9.8 Hz, 1H), 4.16 (t, J=10.1 Hz, 1H), 3.98 (q, J=11.0 Hz, 1H), 3.86 (d, J=11.9 Hz, 1H), 3.72-3.69 (m, 1H), 3.10-3.06 (m, 1H), 3.00-2.82 (m, 1H), 2.74-2.70 (m, 1H), 1.06 (dd, J=27.2, 6.8 Hz, 6H). ³¹P-NMR (162 MHz, DMSO-d₆): δ 53.92 (s, 1P), 52.99 (s, 1P).

The second fractions (T_(R): 10.00 min) gave (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl) amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-B). LCMS (ES, m/z): 866.7 [M+H]⁺.

The third fractions (T_(R): 11.27-12.16 min) gave (5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-C), a mixture of two diastereomers, which was used in next step. LCMS (ES, m/z): 866.7 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.77 (s, 1H), 8.74 (s, 1H), 8.72 (s, 1H), 8.12-8.02 (m, 2H), 7.66-7.64 (m, 1H), 7.56 (t, J=7.5 Hz, 2H), 6.47-6.44 (m, 1H), 5.99 (d, J=8.6 Hz, 1H), 5.55-5.33 (m, 2H), 5.22 (d, J=11.6 Hz, 1H), 4.47 (d, J=25.7 Hz, 1H), 4.43-4.40 (m, 1H), 4.03-3.98 (m, 2H), 3.84 (d, J=11.8 Hz, 1H), 3.75-3.72 (m, 1H), 3.18-3.15 (m, 1H), 2.82-2.73 (m, 2H), 1.13 (dd, J=6.9, 2.5 Hz, 6H). ³¹P-NMR (162 MHz, DMSO): δ 53.42 (s, 1P), 52.16 (s, 1P).

Step 7: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1 and 2)

(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-C) (180 mg, 0.208 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 5.0 mL, 42 mmol), and the resulting solution was stirred at rt for 1h. The volatile components were removed under reduced pressure to give a crude sample that was purified by Prep-HPLC (Atlantis Prep T3 OBD Column, 19 mm×250 mm) eluted with 5 to 19.5% ACN in aq NH₄HCO₃ (50 mM) over 19 min to give, after concentration:

Example 117 (T_(R): 14.82 min): 2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.14 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 6.37 (t, J=5.5 Hz, 1H), 5.99 (d, J=8.7 Hz, 1H), 5.54 (d, J=3.3 Hz, 0.5H), 5.48-5.30 (m, 1.5H), 5.12 (dd, J=10.2, 5.5 Hz, 1H), 4.66 (d, J=34.1 Hz, 1H), 4.36 (s, 1H), 4.24-4.01 (m, 4H), 3.04 (dt, J=14.1, 5.6 Hz, 1H), 2.79 (dt, J=13.5, 6.4 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −198.66 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 53.97 (s, 1P), 53.46 (s, 1P).

Example 118 (T_(R): 15.93 min): 2-amino-9-[(5R,7R,8 S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.16 (s, 1H), 8.11 (s, 1H), 7.83 (s, 1H), 6.34 (dd, J=6.5, 3.0 Hz, 1H), 5.95 (d, J=8.6 Hz, 1H), 5.69-5.53 (m, 1H), 5.47 (d, J=3.4 Hz, 0.5H), 5.33 (d, J=3.4 Hz, 0.5H), 5.23 (p, J=7.3 Hz, 1H), 4.64 (d, J=26.7 Hz, 1H), 4.35 (ddd, J=10.6, 6.9, 3.2 Hz, 1H), 4.31-4.17 (m, 2H), 4.05-3.95 (m, 2H), 2.94-2.85 (m, 1H), 2.74 (dt, J=14.0, 7.2 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −198.74 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 55.05 (s, 1P), 52.87 (s, 1P).

Example 119: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12_(H)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3)

Step 1: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(5R,7R,8 S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-B) (45 mg, 0.053 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 1.5 mL, 11 mmol), and the resulting solution was stirred at rt for 1h. The volatile components were removed under reduced pressure, and the residue was purified by Prep-HPLC (Atlantis Prep T3 OBD Column, 19 mm×250 mm) eluted with 18 to 19.5% ACN in aq NH₄HCO₃ (50 mM) over 16 min to give the product (T_(R): 11.22 min). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.32 (s, 1H), 8.15 (s, 1H), 7.96 (s, 1H), 6.41 (t, J=5.7 Hz, 1H), 6.00 (d, J=8.6 Hz, 1H), 5.56 (dt, J=22.9, 10.4 Hz, 1H), 5.40-5.30 (m, 1.5H), 5.19 (d, J=3.6 Hz, 0.5H), 4.64 (d, J=28.3 Hz, 1H), 4.40-4.27 (m, 2H), 4.27-4.17 (m, 1H), 4.02 (d, J=11.9 Hz, 1H), 3.95-3.85 (m, 1H), 2.92 (dt, J=14.1, 5.6 Hz, 1H), 2.79 (td, J=13.8, 13.1, 6.1 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −198.02 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 57.89 (s, 1P), 55.05 (s, 1P).

Example 120: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12_(H)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4)

Step 1: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-disulfide (28-6-A) (45 mg, 0.053 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 1.5 mL, 11 mmol), and the resulting solution was stirred at rt for 1h. The volatile components were removed under reduced pressure, and the residue was purified by Prep-HPLC (Atlantis Prep T3 OBD Column, 19 mm×250 mm) eluted with 4 to 11% ACN in aq NH₄HCO₃ (50 mM) over 17 min to give the product (T_(R): 11.72 min). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.33 (s, 1H), 8.29 (s, 1H), 8.16 (s, 1H), 6.41 (t, J=6.5 Hz, 1H), 6.05 (d, J=8.6 Hz, 1H), 5.49-5.32 (m, 1.5H), 5.23 (d, J=3.5 Hz, 0.5H), 4.79-4.73 (m, 1H), 4.69-4.59 (m, 1H), 4.40-4.32 (m, 1H), 4.23 (q, J=8.9, 7.5 Hz, 2H), 4.07 (d, J=11.8 Hz, 1H), 3.94-3.84 (m, 1H), 3.00 (dt, J=12.7, 6.2 Hz, 1H), 2.94-2.84 (m, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −197.92 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 59.46 (s, 1P), 54.42 (s, 1P).

Example 121, 122, 123: 2-amino-9-[(2R,5S,7R,8R,10R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) and 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 2 and 3)

Step 1. (2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a flask was added (2R,3R,5 S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (4 g, 4.76 mmol), MeCN (23.65 ml), and water (0.158 ml). Pyridine trifluoroacetate (1.104 g, 5.71 mmol) was then added, and the mixture was stirred 1h at rt, after which time tert-butylamine (20.02 ml, 190 mmol) was added. After stirring 1h at rt, the mixture was concentrated under reduced pressure. The resulting mixture was dissolved in DCM (39.9 ml), and then water (0.798 ml) was added, followed by dichloroacetic acid (2.75 ml, 33.3 mmol). The solution was stirred for 20 min at rt, and then triethylsilane (133 ml, 833 mmol) was added, and the reaction was stirred for a further 2h at rt. After cooling to 0° C., pyridine was added, and the mixture was concentrated under reduced pressure. The resulting sample was partitioned between hexanes (100 mL) and water (20 mL). The layers were separated, and the aqueous phase was purified by reverse phase HPLC using a gradient solvent system of 0-35% MeCN in 0.04% aq ammonium bicarbonate. The product-containing fractions were collected and lyophilized to give (2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate. LCMS (ES, m/z): 400 [M−H]⁻.

Step 2: (2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl) (phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

To a flask was added N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (4 g, 6.09 mmol), DCM (71.7 ml), and 4,5-dicyanoimidazole (2.158 g, 18.27 mmol), and the solution was cooled to 0° C. 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (6.77 ml, 21.32 mmol) was added, and the mixture was stirred for 15 min at 0° C., after which time the mixture was concentrated under reduced pressure and purified by silica gel chromatography using a gradient of 30-100% EtOAc in hexanes to yield (2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite. LCMS (ES, m/z): 857 [M+H]⁻.

Step 3: (2R,3R,5S)-5-((((((2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a flask containing (2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (1.62 g, 2.81 mmol) was added pyridine trifluoroacetate (0.543 g, 2.81 mmol), activated 4 Å sieves and MeCN (10 mL), and the mixture was stirred 20 min at rt. To a separate flask containing (2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2.408 g, 2.81 mmol) was added MeCN (10 mL) and activated 4 Å sieves, and the mixture was stirred 20 min at rt, after which time the hydrogen phosphonate solution was added, and MeCN (2×4 mL) was used to complete the transfer. The mixture was stirred 1h at rt, and then ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione (0.634 g, 3.09 mmol) was added. The resulting mixture was stirred 30 min at rt and then concentrated under reduced pressure. Reverse phase HPLC purification using a gradient solvent system of 5-100% MeCN in 0.04% aqueous ammonium bicarbonate yielded (2R,3R,5S)-5-((((((2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy) phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate. LCMS (ES, m/z): 1187 [M−H]⁻.

Step 4: N-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide

To a flask containing (2R,3R,5 S)-5-((((((2R,3 S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl hydrogen phosphonate (1.34 g, 1.127 mmol) was added DCM (22.54 ml) and water (0.203 ml, 11.27 mmol), and then dichloroacetic acid (1.116 ml, 13.52 mmol) was added. The solution was stirred for 20 min at rt, and then triethylsilane (28.1 ml, 176 mmol) was added. After stirring 3h at rt, the mixture was concentrated under reduced pressure. Pyridine (50 mL) was added, and then the mixture was concentrated under reduced pressure. This process was repeated 2×, and then pyridine (37.6 ml) was added, followed by 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (0.624 g, 3.38 mmol). The resulting mixture was stirred for 1h at rt, after which time water (610l, 33.8 mmol) was added, followed by 3H-1,2-benzodithiol-3-one (285 mg, 1.692 mmol). The resulting mixture was stirred 30 min at rt and then concentrated under reduced pressure. HPLC purification using a gradient solvent system of MeCN in 100 mM aqueous triethylammonium acetate yielded 3 separate diastereomers of N-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl})-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide after lyopholization, all of which showed LCMS (ES, m/z): 899 [M−H]*.

Step 5: 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To a vial containing one diastereomer (slowest eluting) of N-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide (122 mg, 0.135 mmol) was added methylamine (33% in Et0H) (2 mL, 16.07 mmol) and the resulting solution was stirred for 4h at rt, after which time it was concentrated under reduced pressure. Purification by reverse phase HPLC using a gradient solvent system of MeCN in 100 mM aqueous triethylammonium acetate yielded Example 121, 2-amino-9-[(2R,5S,7R,8R,10R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1). LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆) δ 10.54 (s, 1H), 8.01 (s, 1H), 7.86 (s, 1H), 7.29 (d, J=2.7 Hz, 1H), 6.97 (s, 2H), 6.60-6.53 (m, 3H), 6.44 (t, J=6.9 Hz, 1H), 5.73 (d, J=2.7 Hz, 1H), 4.95 (s, 1H), 4.80 (s, 1H), 4.38-4.27 (m, 2H), 3.97-3.91 (m, 2H), 3.90-3.82 (m, 1H), 3.64 (dt, J=16.1, 9.3 Hz, 1H), 2.69-2.63 (m, 1H), 2.63 (s, 12H), 2.60-2.54 (m, 3H), 2.22-2.13 (m, 1H), 1.01-0.94 (m, 18H).

The other diastereomers from Step 4 were treated in an analogous manner to produce two additional diastereomers of 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one:

Example 122 (Diastereomer 2): LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆) δ 10.57 (s, 1H), 9.55 (s, 2H), 8.10 (s, 1H), 7.95 (s, 1H), 7.47 (s, 1H), 7.42 (d, J=3.2 Hz, 1H), 6.66 (d, J=3.4 Hz, 1H), 6.51 (s, 2H), 6.49-6.44 (m, 1H), 5.73 (d, J=4.0 Hz, 1H), 5.08-5.00 (m, 2H), 4.31-4.27 (m, 1H), 4.22 (t, J=11.3 Hz, 1H), 4.07 (q, J=10.6 Hz, 1H), 4.00-3.95 (m, 1H), 3.76-3.71 (m, 1H), 3.65 (td, J=11.6, 6.1 Hz, 1H), 3.02 (d, J=4.2 Hz, 12H), 2.67-2.61 (m, 1H), 2.61-2.53 (m, 2H), 2.18 (dt, J=11.7, 5.3 Hz, 1H), 1.12 (t, J=7.3 Hz, 18H).

Example 123 (Diastereomer 3): LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆) δ 10.53 (s, 1H), 8.01 (s, 3H), 7.28 (d, J=3.4 Hz, 1H), 6.97 (s, 2H), 6.56 (d, J=3.5 Hz, 1H), 6.49 (s, 2H), 6.44 (dd, J=8.6, 5.8 Hz, 1H), 5.70 (d, J=3.9 Hz, 1H), 5.00-4.92 (m, 1H), 4.90-4.83 (m, 1H), 4.33-4.27 (m, 1H), 4.12-3.96 (m, 4H), 3.81 (q, J=12.8, 11.6 Hz, 1H), 3.69 (dd, J=12.0, 6.0 Hz, 2H), 2.83-2.77 (m, 1H), 2.65 (d, J=37.6 Hz, 12H), 2.23 (dt, J=12.8, 6.7 Hz, 1H), 1.03-0.92 (m, 18H).

Examples 124, 125, and 126: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1-3)

Step 1: (2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phenyl phosphonate

To a solution of N-(9-((2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (630 mg, 0.96 mmol) in pyridine (5 mL) under Ar was added diphenyl phosphonate (1.07 g, 4.56 mmol), and the mixture was stirred at rt for 20 min. It was used for the next reaction step without purification. LCMS (ES, m/z): 798.3 [M+H]⁺.

Step 2: t (2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the reaction mixture from Step 1 at 0° C. was added water (1 mL), triethylamine (1 mL).

The resulting mixture was stirred at rt for 20 min. Then, it was concentrated, and the residue was partitioned between CH₂Cl₂ (50 mL) and aq NaHCO₃ (5%, 20 mL). The organic layer was washed with aq NaHCO₃ (5%, 20 mL), dried (Na₂S₂O₄), concentrated and purified by silica gel column chromatography using 0-7% MeOH in CH₂Cl₂ (1% Et₃N) to give the product. LCMS (ES, m/z): 722.2 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 7.76 (s, 1H), 7.74 (s, 0.5H), 7.51 (d, J=1.5 Hz, 1H), 7.48 (q, J=2.4, 1.9 Hz, 1H), 7.41-7.34 (m, 4H), 7.34-7.27 (m, 2H), 7.26-7.21 (m, 1H), 6.92-6.85 (m, 4H), 6.22 (s, 1H), 6.15 (s, 0.5H), 5.37 (d, J=2.7 Hz, 0.5H), 5.28-5.19 (m, 1.5H), 4.73-4.69 (m, 0.5H), 4.66-4.62 (m, 1H), 3.80 (s, 6H), 3.65-3.55 (m, 1H), 3.53-3.44 (m, 1H), 3.12 (q, J=7.3 Hz, 8H), 2.75 (p, J=6.8 Hz, 1H), 1.33-1.22 (m, 18H). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.67 (s, 1P).

Step 3: (2R,3S,4S,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a stirred solution of (2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (540 mg, 0.64 mmol) in CH₂Cl₂ (7 mL) at rt was added water (0.115 g, 6.4 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (6%, 7 mL, 5.76 mmol). The mixture was stirred at rt for 15 min, and then Et₃SiH (15 mL) was added. After 40 min, pyridine (0.90 mL) was added, and the mixture was stirred for 5 min. It was concentrated, and the residue was used for next reaction step without purification. LCMS (ES, m/z): 419.9 [M+H]⁺.

Step 4. (2R,3S,4S,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

The crude from Step 3 was co-evaporated with ACN (3×5 mL), re-dissolved in ACN (3 mL) under Ar, and dried by adding activated 4 Å molecular sieve (100 mg). (1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl (2-cyanoethyl) diisopropylphosphoramidite (684.84 mg, 0.774 mmol) was co-evaporated with ACN (3×5 mL), re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecular sieve (100 mg). After 30 min, it was added to the previously prepared mixture containing pyridin-1-ium (2R,3 S,4S,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate. The resulting mixture was stirred at rt for 30 min. Then it was used in the next reaction step without purification. LCMS (ES, m/z): 1202.1 [M+H]⁺.

Step 5. (2R,3S,4S,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To the mixture from Step 4 was added (E)-N,N-dimethyl-N′(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (144.32 mg, 0.704 mmol), and the mixture was stirred at rt for 30 min. Then, it was concentrated, and the crude was used for the next step without purification. LCMS (ES, m/z): 1234.3 [M+H]⁺.

Step 6. (2R,3S,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a solution of the crude from Step 5 in CH₂Cl₂ (7 mL) at rt was added water (120 mg, 6.4 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (6%, 7 mL, 6 mmol). After 5 min, triethylsilane (15 mL) was added, and it was stirred for additional 2h. Then, pyridine (0.9 mL) was added, and it was concentrated. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 933.9 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 8.74-8.64 (m, 1H), 8.58-8.52 (m, 1H), 8.16-8.06 (m, 2H), 7.98-7.88 (m, 1H), 7.75 (d, J=4.6 Hz, 0.5H), 7.67 (t, J=7.3 Hz, 1H), 7.60-7.56 (m, 2H), 6.25-6.22 (m, 1H), 6.19-6.10 (m, 1H), 5.91 (s, 0.5H), 5.46-5.22 (m, 3H), 5.12 (d, J=12.4 Hz, 1H), 4.84-4.49 (m, 3H), 4.35 (tdd, J=13.2, 6.1, 3.0 Hz, 2H), 4.14 (d, J=8.5 Hz, 1H), 4.11-3.96 (m, 3H), 2.91 (dt, J=16.4, 6.0 Hz, 2H), 2.81-2.68 (m, 1H), 1.29-1.21 (m, 6H). ³¹P-NMR: (162 MHz, CD₃OD): δ 67.84, 66.33 (2 s, 1P); 2.65, 2.52 (2 s, 1P).

Step 7: (5R,7R,8S,12aR,14R,15R,15aS,18S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10 (12H)-olate 2-sulfide

To pyridine (30 mL) at −40° C. under Ar was added diphenyl phosphorochloridate (1783.7 mg, 6.64 mmol) and then, a solution of (2R,3 S,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (310 mg, 0.33 mmol, co-evaporated with pyridine 3×5 mL) in CH₂Cl₂ (30 mL) over 20 min. The resulting mixture was stirred at −40° C. for 20 min. It was used in the next step immediately without purification. LCMS (ES, m/z): 916.1 [M+H]⁺.

Step 8. (5R,7R,8S,12aR,14R,15R,15aS,18S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2,10-disulfide

To the solution from Step 7 at −40° C. was added 3H-benzo[c][1,2]dithiol-3-one (83.6 mg, 0.498 mmol) and water (179 mg, 9.92 mmol). The mixture was stirred at rt for 40 min. Then, it was concentrated, and the residue was purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 947.8 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 8.79-8.58 (m, 2H), 8.18-8.06 (m, 2H), 7.92 (d, J=13.5 Hz, 1H), 7.69-7.66 (m, 1H), 7.63-7.54 (m, 2H), 6.40-6.14 (m, 2H), 6.03-5.56 (m, 1.5H), 5.39-5.10 (m, 2.5H), 4.93-4.85 (m, 2H), 4.84-4.43 (m, 3H), 4.43-3.98 (m, 3H), 2.95 (t, J=5.9 Hz, 1H), 2.84-2.66 (m, 2H), 1.30-1.19 (m, 6H). ³¹P-NMR (162 MHz, CD₃OD): δ 66.60-64.98 (m, 1P), 56.95-55.65 (m, 1P).

Step 9: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7 (12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomers 13)

(5R,7R,8S,12aR,14R,15R,15aS,18 S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2,10-disulfide (260 mg, 0.27 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 20 mL), and the resulting solution was stirred at rt for 3h. Then, it was concentrated, and the residue was purified by Prep-HPLC (Atlantis Prep RP C18 OBD Column, 19 mm×250 mm) eluted with 0 to 14% ACN in aq NH₄HCO₃ (50 mM) over 25 min to afford the diastereomers of 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one:

Example 124 (T_(R): 22.52 min): LCMS (ES, m/z): 719.0 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O+DCl): δ 8.71 (s, 1H), 8.26 (s, 1H), 8.09 (s, 1H), 5.73 (s, 1H), 5.65 (s, 1H), 5.22-5.10 (m, 1H), 4.74-4.72 (m, 2H), 4.45 (d, J=4.1 Hz, 1H), 4.21-4.11 (m, 1H), 4.05-3.94 (m, 2H), 3.83-3.56 (m, 2H), 3.46 (s, 2H). ³¹P-NMR: (162 MHz, D₂O+DCl): δ 60.35 (s, 1P), 56.87 (s, 1P).

Example 125 (T_(R): 15.75 min): LCMS (ES, m/z): 719.0 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O): δ 8.31 (s, 1H), 8.14 (s, 1H), 7.74 (s, 1H), 6.11 (s, 1H), 6.06 (s, 1H), 5.61-5.49 (m, 1H), 5.35 (s, 1H), 5.10 (d, J=9.8 Hz, 1H), 4.71-4.55 (m, 1H), 4.51-4.20 (m, 3H), 4.18-3.95 (in, 4H). ³P-NMR: (162 MHz, D₂O): δ 54.87-51.81 (mn, 2P).

Example 126 (T_(R): 13.17 min): LCMS (ES, m/z): 718.8 [M−H]−. ¹H-NMR: (300 MHz, D₂O+DCl): δ 8.89 (s, 1H), 8.46 (s, 1H), 8.29 (s, 1H), 5.95 (s, 1H), 5.88 (s, 1H), 5.30-5.14 (mn, 1H), 4.98 (t, J=5.2 Hz, 1H), 4.89 (s, 1H), 4.60 (s, 1H), 4.35-4.14 (m, 2H), 4.07 (d, J=11.4 Hz, 1H), 4.01-3.85 (m, 2H), 3.68-3.62 (in, 2H). ³P-NM/R: (121 MHz, D₂O+DCl): δ 60.15 (s, 1P), 56.60 (s, 1P).

Examples 127 and 128: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1), and 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H1-purin-6-one (Diastereomer 2)

Step 1. (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl phenyl phosphonate

To a stirred solution of N-(3-2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide (770 mg, 1.138 mmol) in pyridine (5 ml) at 0° C. under Ar was added diphenyl phosphonate (1.33 g, 5.69 mmol) over 2 min. The resulting mixture was stirred at rt for 20 min. It was used for the next reaction step directly without purification.

Step 2: (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate

To the reaction mixture from Step 1 at 0° C. was added Et₃N (0.56 mL) in water (0.56 mL) over 5 min. The resulting mixture was stirred at rt for 30 min. It was concentrated, and the residue was partitioned between CH₂Cl₂ (60 mL) and aq NaHCO₃ (5%, 24 mL). The organic layer was washed with aq NaHCO₃ (5%, 2×24 mL), dried (Na₂SO₄), concentrated, and purified by chromatography on silica gel using 0-10% MeOH in CH₂Cl₂ (0.5% Et₃N) to give the product. LCMS (ES, m/z): 741.2 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 8.79 (s, 1H), 8.21-8.11 (m, 2H), 7.71 (t, J=7.4 Hz, 1H), 7.67-7.58 (m, 2.5H), 7.46-7.37 (m, 2H), 7.30-7.23 (m, 4H), 7.18-7.13 (m, 2H), 7.03 (dd, J=6.4, 2.8 Hz, 1H), 6.76-6.73 (m, 2H), 6.69-6.65 (m, 2H), 6.04 (s, 0.5H), 5.85 (t, J=6.4 Hz, 0.5H), 5.72 (t, J=6.4 Hz, 0.5H), 5.60 (td, J=16.9, 6.9 Hz, 1H), 4.44-4.40 (m, 1H), 3.77-3.68 (m, 8H), 3.57 (dd, J=10.6, 2.9 Hz, 1H), 2.91 (q, J=7.3 Hz, 18H), 1.20 (t, J=7.3 Hz, 27H). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.48 (s, 1P).

Step 3: (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl phosphonate

To a solution of (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (610 mg, 0.65 mmol) in CH₂Cl₂ (9.8 mL) was added water (0.12 g, 6.5 mmol) and dichloroacetic acid in CH₂Cl₂ (0.6M, 9.8 mL, 5.9 mmol). After 30 in, triethylsilane (20 mL) was added and stirring was continued for additional 2h. Then, pyridine (5 mL) was added, and it was concentrated. The crude was used for the next reaction step without purification. LCMS (ES, m/z): 439.1 [M+H]⁺.

Step 4: (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphanyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate

The crude product of Step 3 was co-evaporated with dry ACN (3×3 mL), re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecular sieve (200 mg). (2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.824 g, 0.960 mmol) was co-evaporated with dry ACN (3×3 mL), re-dissolved in ACN (5 mL), and dried by adding activated 4 Å molecular sieve (200 mg). After 30 min, it was added to the previously prepared mixture containing pyridin-1-ium (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl phosphonate. It was stirred at rt for 30 min, and the reaction mixture was used in the next reaction step directly without purification. LCMS (ES, m/z): 893.2 [M+H]⁺.

Step 5: Diastereomers (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (116-5-A) and (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (116-5-B)

To the reaction mixture from Step 4 at rt, was added (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (181 mg, 0.880 mmol), and the mixture was stirred for 1h. Then, it was concentrated, and the residue was purified by reverse phase (AQ-C18) chromatography eluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) over 88 min.

The first fractions with desired mass (T_(R)=50 min) gave (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (116-5-A). LCMS (ES, m/z): 924.9 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD): δ 8.90 (s, 1H), 8.31 (s, 1H), 8.10 (d, J=7.8 Hz, 2H), 7.75 (s, 0.5H), 7.69 (t, J=7.4 Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 7.02 (dd, J=6.5, 2.6 Hz, 1H), 6.27-6.13 (m, 1.5H), 5.79-5.51 (m, 3H), 5.47-5.21 (m, 12H), 4.58 (dd, J=15.4, 7.6 Hz, 2H), 4.50-4.25 (m, 3H), 4.05 (ddd, J=24.7, 13.2, 6.1 Hz, 2H), 3.90-3.76 (m, 4H), 2.85-2.56 (m, 3H), 1.34 (d, J=6.5 Hz, 3H), 1.25-1.19 (m, 3H). ³¹P-NMR (162 MHz, CD₃OD): δ 67.56 (s, 1P), 3.09 (s, 1P).

The second fractions with desired mass (T_(R)=55 min) gave (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (116-5-B). LCMS (ES, m/z): 925.1 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD): δ 8.87 (s, 1H), 8.33 (s, 1H), 8.16 (d, J=7.8 Hz, 2H), 7.75 (s, 0.5H), 7.70 (t, J=7.4 Hz, 1H), 7.61 (t, J=7.7 Hz, 2H), 6.91 (dd, J=6.2, 3.4 Hz, 1H), 6.23 (d, J=7.1 Hz, 1H), 6.17 (s, 0.5H), 5.74-5.54 (m, 3H), 5.46 (d, J=4.6 Hz, 0.5H), 5.33 (d, J=4.6 Hz, 0.5H), 4.65-4.24 (m, 4H), 4.15 (dt, J=13.0, 5.8 Hz, 2H), 3.95-3.75 (m, 2H), 2.90-2.67 (m, 3H), 1.36-1.31 (m, 3H), 1.19 (d, J=6.8 Hz, 3H). ³¹P-NMR: (162 MHz, CD₃OD): δ 67.29 (s, 1P), 3.07 (s, 1P).

Step 6: (5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl)amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate 10-sulfide

To pyridine (10 mL) at −40° C. under Ar was added diphenyl phosphorochloridate (628 mg, 2.34 mmol), and then, a solution of (2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)-oxy)methyl)-4-fluorotetrahydrofuran-3-yl phosphonate (116-5-B) (110 mg, 0.105 mmol, co-evaporated with pyridine 3×5 mL) in CH₂Cl₂ (10 mL) over 20 min. It was stirred at −40° C. for 20 min. The reaction mixture was used in the next step immediately without purification.

Step 7: (5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl)amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate 2,10-disulfide

To the mixture from Step 6 at −20° C. was added 3H-benzo[c][1,2]dithiol-3-one (27.8 mg, 0.165 mmol) and water (42 mg, 2.3 mmol). The resulting mixture was stirred at rt for 30 min. It was concentrated, and the residue was purified by reverse phase (AQ-C18) chromatography eluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 938.9 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD): δ 8.94-8.71 (m, 2H), 8.23-8.06 (m, 2H), 7.75-7.67 (m, 1H), 7.64 (t, J=7.5 Hz, 2H), 7.11-6.97 (m, 1H), 6.32-6.17 (m, 2H), 6.02-5.73 (m, 2H), 5.68-5.38 (m, 1H), 4.73 (d, J=24.3 Hz, 1H), 4.68-4.42 (m, 4H), 4.39-4.19 (m, 2H), 4.06-3.66 (m, 2H), 2.99-2.62 (m, 2H), 2.61-2.37 (m, 1H), 1.20 (d, J=6.8 Hz, 3H), 1.12 (d, J=6.7 Hz, 3H). ³¹P-NMR: (162 MHz, CD₃OD): δ 64.05, 63.79 (2 s, 1P); 56.67, 56.27 (2 s, 1P).

Step 8: 2-amino-9-(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1, 2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1 and 2)

To pyridinium (5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl) amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate 2,10-disulfide in a steel tank (150 mL) at −60° C. was added ammonia in isopropanol (50 mL) at −60° C. The reactor was tightly sealed and then, heated at 50° C. for 16h. Then, the volatile components were removed under reduced pressure, and the residue was purified by prep-HPLC (Atlantis Prep T3 OBD Column, 19×250 mm) eluted with 0 to 5% ACN in aq NH₄HCO₃ (50 mM) over 25 min.

Example 127 (T_(R)=17.32 min): 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1). LCMS (ES, m/z): 709.8 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O): δ 8.32 (s, 1H), 8.27 (s, 1H), 6.78 (dd, J=11.1, 4.9 Hz, 1H), 6.07 (d, J=8.6 Hz, 1H), 5.81 (t, J=4.6 Hz, 0.5H), 5.68 (t, J=4.7 Hz, 0.5H), 5.63 (dq, J=9.8, 5.0 Hz, 1H), 5.58 (d, J=3.5 Hz, 0.5H), 5.45 (d, J=3.4 Hz, 0.5H), 5.43-5.26 (m, 1H), 4.65-4.63 (m, 1H), 4.56 (q, J=5.6 Hz, 1H), 4.31-4.23 (m, 3H), 4.12-4.05 (m, 1H). ³¹P-NMR: (162 MHz, D₂O): δ 55.76 (s, 1P), 54.26 (s, 1P).

Example 128 (T_(R)=21.10 min): 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2). LCMS (ES, m/z): 709.8 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O): δ 8.26 (s, 1H), 8.01 (s, 1H), 6.79 (dd, J=9.4, 5.3 Hz, 1H), 6.05 (d, J=8.6 Hz, 1H), 5.85 (t, J=5.0 Hz, 0.5H), 5.72 (t, J=5.0 Hz, 0.5H), 5.65-5.35 (m, 3H), 4.69-4.62 (m, 1H), 4.48 (d, J=5.5 Hz, 1H), 4.42 (t, J=11.1 Hz, 1H), 4.18 (t, J=6.2 Hz, 2H), 4.09-4.06 (m, 1H). ³¹P-NMR (162 MHz, D₂O): δ 54.74 (s, 1P), 53.84 (s, 1P).

Examples 129 through 243, as shown in Table 6 below, were or may be prepared according to procedures analogous to those outlined in Examples 116 through 128 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.

TABLE 6 Mass Ex. Structure Name [M − H]⁻ 129

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra- decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 710 130

2-amino-9- [(2R,5S,7R,8R,10R,12aR,14R,15S,15aR)-14-(6- amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 691 131

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)- 14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 691 132

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)- 14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-oe (Diastereomer 3) 691 133

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 694 134

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 719 135

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 719 136

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 719 137

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 4) 719 138

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 692 139

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dioxido-2,10- disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 701 140

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-2,10-dioxido-2,10- disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 701 141

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dioxido-2,10- disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diasteroemer 3) 701 142

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dioxido-2,10- disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 4) 701 143

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-10-oxido-2-sulfidooctahydro-12H- 5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 694 144

2-amino-9- [(2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16S)- 14-(6-amino-9H-purin-9-yl)-15,16-difluoro- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 709 145

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 709 146

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 709 147

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra- decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 148

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra- decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 710 149

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 673 150

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 673 151

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15aS)-14-(4-aminoimidazo[2,1- f][1,2,4]triazin-7-yl)-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 673 152

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 4) 673 153

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6- amino-9H-purin-9-yl)-15,16-difluorooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecine-2,10-bis(thiolate) 2,10- dioxide (Diastereomer 1) 725 154

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6- amino-9H-purin-9-yl)-15,16-difluorooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (Diastereomer 2) 725 155

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-15,16,16-trifluoro- 2,10-dihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 727 156

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-15,16,16-trifluoro- 2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 727 157

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-18,18-difluoro-2,10- dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 737 158

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-18,18-difluoro-2,10- dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 737 159

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-18,18-difluoro-2,10- dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3) 737 160

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-18,18-difluoro-2,10- dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 4) 737 161

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 690 162

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 690 163

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3) 690 164

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 4) 690 165

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 728 166

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 728 167

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 3) 728 168

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 708 169

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 708 170

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 3) 708 171

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15S,15aR)-14-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 690 172

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 690 173

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15S,15aR,16S)-14-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro- 2,10-dihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1) 708 174

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 708 175

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 692 176

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 692 177

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 1) 710 178

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 2) 710 179

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 3) 710 180

1-[(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,5-dihydro-4H- imidazo[4,5-c]pyridin-4-one 693 181

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 693 182

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo- tetradecin-7-yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 693 183

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 693 184

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 702 185

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 702 186

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6- dihydro-7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 3) 702 187

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14- (6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 4) 702 188

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 673 189

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 673 190

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 673 191

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 192

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15-fluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 193

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 706 194

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 1) 691 195

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 691 196

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 3) 691 197

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 710 198

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 2) 710 199

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 710 200

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 711 201

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 711 202

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one 711 203

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 720 204

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 720 205

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 720 206

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one 709 207

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)- 14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 716 208

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)- 14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 2) 716 209

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)- 14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo- tetradecin-7-yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 716 210

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 692 211

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 692 212

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 692 213

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 692 214

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 1) 725 215

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 2) 725 216

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3) 725 217

2-amino-9- [(2R,5R,7R,8R,10R,12aR,14R,15S,15aR,16S)- 14-(6-amino-9H-purin-9-yl)-15,16-difluoro- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 4) 725 218

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 1) 726 219

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 726 220

5-amino-3-[(5R,7R,8S,12aR,14R,15aR,16S)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 15,15,16-trifluoro-2,10-dihydroxy-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one 727 221

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 1) 726 222

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 2) 726 223

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 726 224

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 1) 727 225

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 727 226

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 1) 725 227

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 2) 725 228

1-[(5S,7R,8R,12aR,14R,15S,15aR)- 14-(4-amino-7H-pyrrolo[2,3-d] pyrimidin-7-yl)-15-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one 674 229

6-amino-1- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,5-dihydro- 4H-imidazo[4,5-c]pyridin-4-one (Diastereomer 1) 707 230

6-amino-1- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16- difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,5-dihydro-4H- imidazo[4,5-c]pyridin-4-one (Diastereomer 2) 707 231

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 726 232

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 726 233

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 726 234

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 706 235

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6- amino-9H-purin-9-yl)-15,16-difluoro-2,10- dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one 726 236

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 744 237

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 2) 744 238

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 744 239

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 4) 744 240

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 735 241

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l] [1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 735 242

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6- amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido- 2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3) 735 243

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7- amino-3H-imidazo[4,5-b]pyridin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one 724

Examples 244, 245, 246, and 247: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,1-dihydroxy-2,1-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1-3) and 2-amino-9-1(2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4)

Step 1. (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

Pyrrole (0.087 mL, 1.2 mmol) was added to a solution of (2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate triethylamine salt (1:2) (0.34 g, 0.41 mmol) in acetonitrile (3.0 mL) under an argon atmosphere at 0° C. After 5 min, TFA (0.096 mL, 0.14 mmol) was added, and the reaction mixture was stirred at 0° C. for 30 min. Pyridine (0.13 mL, 1.7 mmol) was added drop wise at 0° C. The reaction mixture was then stirred for 10 min at 0° C. At that time, a mixture of (2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.48 g, 0.55 mmol) in acetonitrile (3.0 mL) was added drop wise over 5 min to the reaction mixture under an argon atmosphere at 0° C. The reaction mixture was stirred at 0° C. for 20 min and immediately used in the next step without further manipulation.

Step 2: (2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To the crude reaction mixture from Step 1 was added (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (0.10 g, 0.50 mmol) under an argon atmosphere at 0° C. The reaction mixture was stirred for 45 minutes at 0° C. At that time, 1-propanol (0.31 mL, 4.13 mmol) was added to the reaction mixture under an argon atmosphere at 0° C. The reaction mixture was then allowed to warm to ambient temperature and stirred for 10 min. TFA (0.32 mL, 4.1 mmol) was added to the reaction mixture, and the reaction mixture was stirred for 30 min at ambient temperature. Pyridine (0.37 mL, 4.6 mmol) was added at ambient temperature, and the reaction mixture was stirred for 10 min. The reaction mixture was concentrated under reduced pressure to approximately one-half volume. The mixture was then diluted with isopropyl acetate (20 mL) and stirred for 30 min at ambient temperature. The resulting suspension was filtered.

The collected solids were dried overnight under high vacuum to afford (2R,3 S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate. LCMS (ES, m/z): 922 [M−H]⁻.

Step 3: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(2R,3 S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)-methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (0.30 g, 0.33 mmol) was azeotroped with dry pyridine (2×10 mL) and then dried under high vacuum for 1 h. In a separate flask, diphenyl phosphorochloridate (0.34 mL, 1.6 mmol) was added to a mixture of acetonitrile (15 mL) and pyridine (1.0 mL). The resulting solution was then cooled to −20° C. To this mixture was added drop wise over a period of 5 min a mixture of (2R,3 S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)-methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (0.30 g, 0.33 mmol) in pyridine (4.0 mL) at −20° C. The reaction mixture was then stirred at −20° C. for 15 min post-addition. 3H-benzo[c][1,2]dithiol-3-one (0.066 g, 0.39 mmol) and water (0.12 mL, 6.5 mmol) were then added to the reaction mixture at −20° C. The reaction mixture was allowed to gradually warm to ambient temperature. The reaction mixture was stirred for 30 min at ambient temperature. The reaction mixture was then concentrated under reduced pressure to approximately one quarter volume. The reaction mixture was cooled to 0° C., and methanamine (33% in ethanol) (2.63 mL, 24 mmol) was added drop wise. After the addition was complete, the reaction mixture was allowed to warm to ambient temperature. The reaction mixture was stirred at ambient temperature for 18h. The reaction mixture was concentrated under reduced pressure to afford the crude product residue. The crude product residue was azeotroped (3×30 mL ethanol) to afford the crude product. This material was dissolved in water (5 mL) and acetonitrile (1 mL). The resulting mixture was purified by mass-directed reverse phase HPLC (Waters Sunfire 19×250 mm, UV 215/254 nm, fraction trigger by SIM negative MS monitoring mass 709; mobile phase=100 mM triethylammonium acetate in water/acetonitrile gradient, 2-30% acetonitrile over 40 min) to afford the 4 diastereomers of 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15 aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one.

Example 244: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1): T_(R)=17.7 min. LCMS (ES, m/z): 709 [M−H]⁻.

Example 245: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2): T_(R)=21.9 min. LCMS (ES, m/z): 709 [M−H]⁻. 1H NMR (500 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.21-8.09 (m, 2H), 7.46-7.29 (m, 2H), 6.59-6.43 (m, 2H), 6.40-6.29 (m, 1H), 5.88 (d, J=8.8 Hz, 1H), 5.49-5.19 (m, 4H), 4.45-4.32 (m, 2H), 4.10-3.93 (m, 2H), 3.94-3.82 (m, 1H), 3.80-3.68 (m, 1H).

Example 246: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3): T_(R)=23.8 min. LCMS (ES, m/z): 709 [M−H]⁻. 1H NMR (500 MHz, DMSO-d₆) δ 8.18-8.08 (m, 3H), 7.41-7.33 (m, 2H), 6.59-6.47 (m, 2H), 6.37-6.27 (m, 1H), 5.84 (d, J=8.7 Hz, 1H), 5.52-5.26 (m, 2H), 5.21-5.11 (m, 1H), 4.46-4.35 (m, 2H), 4.19-4.02 (m, 2H), 3.83-3.65 (m, 2H).

Example 247: 2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15S,15 aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4): T_(R)=26.4 min. LCMS (ES, m/z): 709 [M−H]⁻. ¹H NMR (500 MHz, DMSO-d₆) δ 8.19-8.07 (m, 3H), 7.41-7.32 (m, 2H), 6.70-6.50 (m, 2H), 6.40-6.29 (m, 1H), 5.85 (d, J=8.7 Hz, 1H), 5.33-5.25 (m, 2H), 5.23-5.12 (m, 1H), 4.48-4.35 (m, 1H), 4.33-4.24 (m, 1H), 4.09-3.93 (m, 2H), 3.92-3.81 (m, 1H), 3.83-3.70 (m, 1H).

Examples 248 through 256, as shown in Table 7 below, were prepared according to procedures analogous to those outlined in Examples 244 through 247 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.

TABLE 7 Mass Ex. Structure Name [M − H]⁻ 248

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16- fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 691 249

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)- 14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)- 16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 691 250

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)- 14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)- 16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 691 251

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)- 14-(4-aminoimidazo[2,1-f)[1,2,4]triazin-7-yl)- 16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4) 691 252

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 253

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 254

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 708 255

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-amino-5-fliioro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-16-fluoro-10-hydroxy-2,10-dioxido-2- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 692 256

2-amino-9-[(5R,7R,8S,12aR,4R,15aS,16R)-14- (4-amino-5-fliioro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-16-fluoro-10-hydroxy-2,10-dioxido-2- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 692

Examples 257 through 259 in Table 8 were made according to procedures analogous to those described above for Examples 244 through 247 using the appropriate monomeric nucleosides, described as Preparations or as obtained from commercial sources, with the following additional representative treatment as a final step: A sample (0.12 mmol) was dissolved in water (6 mL), and the resulting mixture was applied to ion exchange resin in a column (DOWEX 50WX2 hydrogen form, 100-200 mesh, 1.5 g, pre-washed with 10 mL water, and then packed in column before compound loading occurred). After loading mixture had completely absorbed to column, the column was then washed with additional water (10 mL). The eluent was lyophilized to afford product.

TABLE 8 Mass Ex. Structure Name [M − H]⁻ 257

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 691 258

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 691 259

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16- fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 691

Alternatively, Examples 117-142, 144-152, 172, 174, and 244-247, above were made using procedures similar to those described for Examples 81 through 83 above.

Example 260: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1. ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethylsilyl)oxy)tetrahydrofuran-2-yl)methyl (2-cyanoethyl) diisopropylphosphoramidite

To a solution of N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (780 mg, 2.089 mmol) in DMF (8 mL) and DIPEA (1.116 mL, 6.39 mmol) at 0° C. was added 200 mg active molecular sieve 4 Å and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (572 mg, 2.343 mmol) in 1 ml dry CH₃CN. The resulting mixture was stirred at 0° C. for 5h; chlorotriethylsilane (401 mg, 2.66 mmol) was added dropwise. The resulting mixture was stirred at rt overnight. The reaction carried directly to next step. LCMS (ES, m/z): 686 [M−H]⁻

Step 2: O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethylsilyl)oxy) tetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-(2-cyanoethyl) phosphorothioate

To a solution of the product of step 1 was added N-(9-((2R,3 S,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (500 mg, 1.065 mmol) and 1H-tetrazole (895 mg, 12.78 mmol). The mixture was stirred at RT for 2h, and DDTT (568 g, 2.77 mmol) was added. The mixture was stirred for 1h, then partitioned between ethyl acetate and H₂O, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel, eluting with 0-6% MeOH/DCM to give O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethyl silyl)oxy)tetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-(2-cyanoethyl) phosphorothioate. LCMS (ES, m/z): 1088 [M+H]⁺

Step 3: O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-hydrogen phosphorothioate

To a solution of the mixture (520 mg) from step 3 in THF (5 mL) was added TBAF (1.0 M in THF) (1.140 mL, 1.140 mmol). The resulting mixture was stirred at RT for 2h and concentrated in vacuo. The residue was purified by column chromatography on silica gel, eluting with 0-8% MeOH/DCM to give O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogen phosphorothioate. LCMS (ES, m/z): 807 [M+H]⁺

Step 4. N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide

O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogen phosphorothioate (250 mg, 0.310 mmol) and diisopropylammonium tetrazolide (80 mg, 0.465 mmol) were azeotrope with dry CH₃CN (3×10 ml) and dried under high vacuum for 30 min.

The above mixture was dissolved in DMF (1 mL) and acetonitrile (7 mL) and added 200 mg active molecular sieve 4 Å and a solution of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (128 mg, 0.403 mmol) in 1 ml dry CH₃CN. The resulting mixture was stirred at rt for 30 min, followed by addition of 1H-tetrazole (109 mg, 1.550 mmol). The reaction stayed at rt for 1h and added DDTT (95 mg, 0.465 mmol). The stirring continued for 1h and concentrated in vacuo. The residue was purified by column chromatography on silica gel, eluting with 0-10% MeOH/DCM with 1% ET3N to give the desired product. LCMS (ES, m/z): 883 [M−H]⁻.

Step 5: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide (120 mg, 0.136 mmol) and ammonia (7.0M in Me0H) (2 ml, 14.00 mmol) were sealed in a microwave tube. The reaction mixture was heated to 50° C. and stirred for 4h. The reaction mixture was concentrated, and purified using mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate to give desired product. Lyopholization of the product fractions furnished 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]-pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one. LCMS (ES, m/z): 709 [M−H]⁻. ¹H NMR (H₂O-d2, 500 MHz): δ_(H) 8.23 (1H, s), 8.17 (1H, s), 7.93 (1H, s), 6.43 (1H, d, J=15.0 Hz), 6.00 (1H, d, J=8.7 Hz), 5.75 (1H, m), 5.58 (2H, m), 5.15 (1H, m), 4.65 (2H, m), 4.54 (1H, m), 4.15-4.30 (3H, m). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 52.1, 52.2.

Examples 261, 262, 263: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1-3)

Step 1: ((2R,3R,4S,5R)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (2-cyanoethyl) diisopropylphosphoramidite

To a solution of N-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (190 mg, 0.535 mmol) and 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (95%) (373 mg, 1.176 mmol) in DMF (5 ml) was added diisopropylammonium tetrazolide (137 mg, 0.802 mmol). The mixture was stirred at RT for 2h, then added 1H-tetrazole (15.77 mg, 0.225 mmol) and continued for 2h. 300 mg active molecular sieve 4 Å was added and continued stirring at RT for 2h. The reaction carried directly to next step. LCMS (ES, m/z): 754 [M−H]⁻

Step 2: N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide

To a solution of N-(7-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (180 mg, 0.454 mmol) and Et₃N (0.114 mL, 0.817 mmol) in DMF (4 mL) at −78° C. was added a solution of TMS-Cl (0.070 mL, 0.545 mmol) in CH₂Cl₂ (1 mL) dropwise. The mixture was stirred at −78° C. for 20 min and warmed to 0° C. within 1h, then added 200 mg active molecular sieve 4 Å and continued stirring at RT for 2h. The mixture was cooled to 0° C. and transferred to a stirred solution of the product of Step 1 (394 mg, 0.454 mmol) (precooled to −0° C.), followed by addition of 1H-tetrazole (191 mg, 2.72 mmol). The mixture was gradually warmed to RT and stirred at RT for 2h. The filtration removed the solids. After washed with 3 ml DMF, the combined filtrate was added additional 1H-tetrazole (191 mg, 2.72 mmol) and stirred at RT overnight.

After completion of reaction, the reaction mixture was diluted with ethyl acetate and washed successively with H₂O and brine. The organic layer was dried over anhydrous Na₂SO₄ and purified by a silica gel chromatography, eluting with 0-8% MeOH/DCM to give two desired fractions with the same molecular weight. LCMS (ES, m/z): 1014 [M+H]⁺

Step 3: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide (faster fraction from step 2) (30 mg, 0.030 mmol) and 7.0M ammonia in MeOH (2 ml, 14.00 mmol) were sealed in a microwave tube. The mixture was heated to 50° C. and stirred for 8h. The reaction mixture was concentrated, and the product was purified using mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate. Lyopholization of the product fractions furnished 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one as the bis-triethylamine salt.

Example 261 (Diastereomer 1): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR (H₂O-d₂, 500 MHz): δ_(H) 8.32 (1H, s), 8.08 (1H, s), 7.05 (1H, s), 6.70 (1H, t, J=6.1 Hz), 6.08 (1H, d, J=8.7 Hz), 5.71 (1H, d, J=53.2 Hz), 5.47-5.40 (1H, m), 5.14-5.10 (1H, m), 4.65 (2H, m), 4.28 (1H, t, J=8.7 Hz), 4.14-4.02 (3H, m), 2.85 (2H, d, J=6.4 Hz). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 53.7, 54.0.

Step 4: 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide (slow fraction from Step 2, 65 mg, 0.064 mmol) and 7.0 M ammonia in MeOH (2 ml, 14.00 mmol) were sealed in a microwave tube and heated to 50° C. and stirred for 8h. The reaction mixture was concentrated and purified using mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate to give two additional diastereomers after lyopholization of the product fractions.

Example 262 (Diastereomer 2): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR (H₂O-d₂, 500 MHz): δ_(H) 8.09 (2H, d, J=3.0 Hz), 7.19 (1H, s), 6.74 (1H, s), 6.08 (1H, d, J=8.3 Hz), 5.63 (1H, s), 5.53 (1H, s), 5.35-5.33 (1H, m), 4.65 (2H, m), 4.34 (1H, d, J=8.8 Hz), 4.28 (1H, t, J=10.3 Hz), 4.11 (1H, d, J=12.1 Hz), 3.89 (1H, dd, J=10.6, 4.0 Hz), 2.85 (2H, d, J=6.4 Hz). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 54.7, 59.8.

Example 263 (Diastereomer 3): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR (H₂O-d₂, 500 MHz): δ_(H) 8.08 (1H, s), 7.94 (1H, s), 7.04 (1H, s), 6.68 (1H, s), 6.04 (1H, d, J=8.6 Hz), 5.64 (1H, s), 5.54 (1H, d, J=3.3 Hz), 5.24 (1H, d, J=7.7 Hz), 4.65 (2H, m), 4.40 (1H, d, J=8.2 Hz), 4.18 (1H, dd, J=11.0, 5.5 Hz), 4.09-4.00 (2H, m), 2.93-2.89 (1H, m), 2.82 (1H, d, J=7.1 Hz). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 53.1, 54.9.

Examples 264 and 265: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) and 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2)

Step 1: (2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

To a solution of (2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate (750 mg, 0.865 mmol) in CH₂Cl₂ (9 mL) was added water (156 mg, 8.65 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6M, 11 mL, 6.6 mmol). The mixture was stirred at rt for 15 min, and then, Et₃SiH (4.00 mL) was added. After 1h, pyridine (1232 mg, 15.57 mmol) was added, and it was concentrated to give a crude sample that was used for next reaction step without purification. LCMS (ES, m/z): 548.1 [M+H]⁺.

Step 2. (2R,3R,4S,5R)-5-((((((R, 3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

The crude sample from Step 1 was co-evaporated with ACN (3×2 mL), re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecular sieve (150 mg). (1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl (2-cyanoethyl) diisopropylphosphoramidite (0.843 g, 0.952 mmol) was also co-evaporated with ACN (3×1 mL), re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecular sieve (150 mg). After 30 min, it was added to the previously prepared mixture containing pyridin-1-ium (2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate. The reaction mixture was used in the next reaction step without purification. LCMS (ES, m/z): 1332.1 [M+H]⁺.

Step 3. (2R,3R,4S,5R)-5-((((((R, 3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

To a reaction mixture from Step 2 at rt was added 2-hydroperoxy-2-methylpropane (0.234 g, 2.60 mmol), and it was stirred for 40 min. It was concentrated to give a crude product, which was used for the next step without purification. LCMS (ES, m/z): 1347.1 [M+H]⁺.

Step 4: (2R,3R,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate

To the crude from Step 3 in CH₂Cl₂ (9 mL) was added water (156 mg, 8.65 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6N, 10 mL, 6 mmol). The resulting mixture was stirred at rt for 15 min. Then, triethylsilane (4 mL, 0.21 mmol) was added, and the stirring was continued for 40 min. Pyridine (1232 mg, 15.57 mmol) was added. The mixture was concentrated, and the residue was purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 1046.3 [M+H]⁺.

Step 5. (5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate 2-oxide

To pyridine (20 mL) at −40° C. under Ar was added diphenyl phosphorochloridate (1.28 g, 4.78 mmol) over 5 min. To the solution at −40° C. was added a solution of (2R,3R,4S,5R)-5-((((((1 S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl) oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl phosphonate (250 mg, 0.239 mmol, co-evaporated with pyridine, 3×10 mL) in CH₂Cl₂ (20 mL) dropwise. The resulting mixture was stirred at −40° C. for 30 min. The reaction mixture was used for the next reaction step immediately without purification. LCMS (ES, m/z): 1028.4 [M+H]⁺.

Step 6: (5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate 2-oxide 10-sulfide

To the reaction mixture at rt from Step 5 was added 3H-benzo[c][1,2]dithiol-3-one (121 mg, 0.717 mmol). After stirring at 25° C. for 40 min, water (431 mg, 23.9 mmol) was added. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse phase (C, 18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 1060.3 [M+H]⁺. ³¹P-NMR: (121 MHz, D₂O) δ 54.91, 54.61 (m, 1P), δ −6.08, −6.48 (m, 1P).

Step 7: (5R,7R,8R,12aR,14R,15R,15aS,18S)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecine-2,10(12H)-diolate 2-oxide 10-sulfide

(5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate 2-oxide 10-sulfide (190 mg, 0.178 mmol) was dissolved in a solution of methylamine in EtOH (30%, 1 mL), and the resulting solution was stirred at rt for 2h. Then, it was concentrated to give a crude sample containing the product. LCMS (ES, m/z): 833.2 [M+H]⁺.

Step 8: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,2a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one

The crude from Step 7 was suspended in pyridine (5 mL) under Ar. To the mixture was added Et₃N (1864 mg, 18.42 mmol) and triethylamine trihydrofluoride (742 mg, 4.60 mmol) dropwise. The mixture was heated to 50° C. for 16h. Then, it was concentrated, and the residue was purified by preparative-HPLC (XBridge Shield RP18 OBD Column, 19×150 mm) eluted with 0 to 14% ACN in aq NH₄HCO₃ (10 mM) over 16 min to give two products after concentration.

Example 264: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1): T_(R): 11.00 min. LCMS (ES, m/z): 719.0 [M+H]⁺. ¹H-NMR: (300 MHz, D₂O): δ 8.13 (s, 1H), 7.94 (s, 1H), 7.64 (s, 1H), 6.11-5.97 (m, 2H), 5.92-5.85 (m, 1H), 5.25-5.05 (m, 1H), 4.87 (s, 1H), 4.75 (s, 2H), 4.30-4.21 (m, 1H), 4.12-3.78 (m, 4H), 3.50-3.45 (m, 1H). ³¹P-NMR: (121 MHz, D₂O) δ 52.06 (s, 1P), δ −0.86 (s, 1P)

Example 265: 2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2): T_(R): 12.32 min. LCMS (ES, m/z): 719.0 [M+H]⁺. ¹H-NMR: (300 MHz, D₂O): δ 8.13-8.03 (m, 2H), 7.64 (s, 1H), 6.11-5.97 (m, 2H), 5.92-5.90 (m, 1H), 5.20-5.15 (m, 1H), 4.87-4.75 (m, 3H), 4.33-4.20 (m, 1H), 4.12-4.00 (m, 2H), 3.90-3.78 (m, 2H), 3.45-3.40 (m, 1H). 31P-NMR: (121 MHz, D₂O) δ 54.88 (s, 1P), δ-0.97 (s, 1P).

Examples 266 through 272, as shown in Table 9 below, were prepared according to procedures analogous to those outlined for Examples 264 and 265 above using the appropriate monomeric nucleosides, described as Preparations or as obtained from commercial sources.

TABLE 9 Mass Ex. Structure Name [M − H]⁻ 266

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16S)- 14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16- trihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one 707 267

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-2,16- dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotctradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 690 268

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-2,16- dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 690 269

5-amino-3-[(5R,7R,8R,12aR,14R,15S,15aR,16S)- 14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15-fluoro-2,16-dihydroxy-2,10-dioxido-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 709 270

5-amino-3-[(5R,7R,8R,12aR,14R,15S,15aR,16S)- 14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15-fluoro-2,16-dihydroxy-2,10-dioxido-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 709 271

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,16- dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 690 272

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14- (4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,16- dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 690

Examples 273 and 274: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H1-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) and 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2)

Step 1. (2R,3,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-, 6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a mixture containing the crude (2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (product of Step 1, Examples 244-247, crude, assumed 0.75 mmol) was added tert-butyl hydroperoxide in decane (5.5M, 0.48 mL, 2.6 mmol) dropwise, and the mixture was stirred at rt for 1h. Then, it was cooled to 0° C., and a solution of Na₂S₂O₃ (553 mg) in water (2 mL) was added slowly. The mixture was stirred at rt for 5 min and then, concentrated to give the product. LCMS (ES, m/z): 1208.5 [M−H]⁻.

Step 2: (2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a solution of the crude from Step 1 in CH₂Cl₂ (10 mL) was added water (130 mg, 7.5 mmol) and 2,2-dichloroacetic acid (0.77 g, 6 mmol) in CH₂Cl₂ (10 mL). After 20 min, triethylsilane (20 mL) was added, and stirring was continued for 2h. Pyridine (1 mL) was added, and the reaction mixture was concentrated. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 908.2 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD) δ 8.76 (d, J=7.7 Hz, 2H), 8.65 (s, 1H), 8.56 (s, 1H), 8.21-8.08 (m, 6H), 7.69 (t, J=7.4 Hz, 2H), 7.64-7.50 (m, 5H), 6.67 (ddd, J=32.7, 14.9, 4.4 Hz, 2H), 6.21-6.10 (m, 2H), 5.99-5.91 (m, 1H), 5.64-5.57 (m, 1H), 5.56-5.45 (m, 3H), 5.38 (s, 3H), 4.71-4.54 (m, 6H), 4.47-4.23 (m, 5H), 3.92 (d, J=3.8 Hz, 1H), 3.89-3.80 (m, 2H), 2.94 (dt, J=31.1, 5.9 Hz, 4H), 2.83-2.74 (m, 2H), 1.36-1.19 (m, 13H), 1.12 (s, 2H). ³¹P-NMR: (162 MHz, CD₃OD) δ 2.55, −1.33, −3.09, −3.11, −155.89.

Step 3. (5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-oxide

To pyridine (50 mL) under Ar was added diphenyl chlorophosphate (2.66 g, 9.92 mmol). It was cooled at −40° C., and then, a solution of (2R,3 S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (450 mg, 496 mmol, co-evaporated with pyridine 3×5 mL) in CH₂Cl₂ (50 mL) was added dropwise over 20 min. The resulting mixture was stirred at −40° C. for 20 min. The reaction mixture was immediately used in the next step without purification. LCMS (ES, m/z): 891.1 [M+H]⁺.

Step 4: (5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-oxide 10-sulfide

To the reaction mixture of Step 3 at −40° C. was added 3H-benzo[c][1,2]dithiol-3-one (32.76 mg, 0.195 mmol) and water (125 mg, 0.744 mmol). After stirring at rt for 40 min, the reaction mixture was concentrated under reduced pressure. The residue was purified by reverse phase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 922.1 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD) δ 8.81-8.73 (m, 2H), 8.63-8.48 (m, 2H), 8.24 (s, 1H), 8.21-8.10 (m, 5H), 7.73-7.56 (m, 7H), 6.83-6.72 (m, 2H), 6.28 (d, J=8.6 Hz, 1H), 6.14 (d, J=8.5 Hz, 1H), 5.83-5.56 (m, 4H), 5.49 (d, J=11.1 Hz, 1H), 4.76-4.46 (m, 11H), 4.37 (s, 1H), 4.29 (d, J=6.8 Hz, 1H), 4.18 (d, J=10.2 Hz, 1H), 3.04 (dd, J=6.5, 5.1 Hz, 4H), 2.83-2.68 (m, 1H), 2.06 (s, 2H), 1.23 (dd, J=22.5, 6.9 Hz, 9H), 1.14-1.05 (m, 3H). ³¹P-NMR: (162 MHz, CD₃OD) δ62.41, 56.84, 56.29, −3.14, −3.35, −4.77, −5.06, −60.84.

Step 5: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomers 1 and 2)

The (5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate 2-oxide 10-sulfide (380 mg, 0.377 mmol) was dissolved in a solution of MeNH₂ in EtOH (30%, 30 mL), and the resulting solution was stirred at rt for 2h. Then, it was concentrated, and the residue was purified by Prep-HPLC (XBridge Shield RP18 OBD Column, 19×150 mm) eluted with 4 to 10% ACN in aq NH₄HCO₃ (10 mM) over 20 min.

Example 273: 2-amino-9-[(5R,7R,8 S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1): T_(R)=10.10 min. LCMS (ES, m/z): 693.0 [M−H]⁻. ¹H-NMR: (400 MHz, CDCl₃) δ 10.84 (d, J=2.4 Hz, 1H), 10.72 (s, 1H), 10.59 (d, J=3.9 Hz, 1H), 9.01 (dd, J=19.4, 3.0 Hz, 1H), 8.56 (d, J=8.5 Hz, 1H), 8.14-7.96 (m, 2H), 7.93-7.82 (m, 1H), 7.73 (dd, J=12.0, 6.5 Hz, 2H), 7.31-7.19 (m, 2H), 7.15 (d, J=2.8 Hz, 1H), 6.94-6.74 (m, 3H), 6.71-6.53 (m, 2H). ³¹P-NMR: (162 MHz, D₂O): δ 62.301 (s, 1P), 0.976 (s, 1P).

Example 274: 2-amino-9-[(5R,7R,8 S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2): T_(R): 14.27 min. LCMS (ES, m/z): 692.9 [M−H]⁻. ¹H-NMR: (400 MHz, DMSO-d6) δ 8.31 (d, J=11.0 Hz, 2H), 8.18 (s, 1H), 6.40 (dd, J=22.6, 2.6 Hz, 1H), 5.88 (d, J=8.8 Hz, 1H), 5.44 (d, J=33.7 Hz, 2H), 5.35-5.23 (m, 1H), 5.24-5.05 (m, 2H), 4.48-4.34 (m, 1H), 4.31 (dd, J=10.6, 4.5 Hz, 1H), 4.16-3.87 (m, 3H). ³¹P-NMR: (162 MHz, DMSO-d6): δ 55.096 (s, 1P), −2.731 (s, 1P).

Examples 275 through 288, as shown in Table 10 below, were prepared according to procedures analogous to those outlined in Examples 274 and 275 above using the appropriate monomeric nucleosides, described as Preparations or as obtained from commercial sources.

TABLE 10 Mass Ex. Structure Name [M − H] 275

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10- dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1) 656 276

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- amino-7H-pyirolo[2,3-d]pyrimidin-7-yl)-2,10- dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2) 656 277

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)- 14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10- dihydroxy-2-oxido-10-sulfidohexahydro-14H- 15,12a-(epoxymethano)-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 703 278

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)- 14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10- dihydroxy-2-oxido-10-sulfidohexahydro-14H- 15,12a-(epoxymethano)-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 703 279

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 14-(7-amino-3H-[1,2,3]triazoIo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10- sulfidooctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 694 280

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10- sulfidooctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 694 281

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15- fluoro-2,10-dihydroxy-2-oxido-10- sulfidooctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 675 282

5-amino-3-[(5R,7R,8S,12aR,14R,15S,15aR,16S)- 14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10- sulfidooctahydro-12H-5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 695 283

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4- aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2- hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H- 5,8-methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotctradecin-7-yl]-1,9- dihydro-6H-purin-6-one 657 284

2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18R)- 14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10- dihydroxy-2-oxido-10-sulfidohexahydro-14H- 15,12a-(epoxymethano)-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7(12H)-yl]-1,9-dihydro-6H-purin-6-one 719 285

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)- 14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin- 3-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10- sulfanyloclahydro-12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one 710 286

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)- 14-(7-amino-3H-iniidazo[4,5-b]pyridin-3-yl)- 15,16-difluoro-2-hydroxy-2,10-dioxido-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 287

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)- 14-(7-amino-3H-imidazo[4,5-b]pyridin-3-yl)- 15,16-difluoro-2-hydroxy-2,10-dioxido-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 288

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14- (7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3- yl)-15,15,16-trifluoro-2-hydroxy-2,10-dioxido-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one 728

Example 289: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1: N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-10-hydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide

O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl) O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogen phosphorothioate (product of Step 3, Example 261, 100 mg, 0.124 mmol) and diisopropylammonium tetrazolide (31.8 mg, 0.186 mmol) were azeotrope with dry CH₃CN (3×10 ml) and dried under high vacuum for 30 min.

The above mixture was dissolved in DMF (1 mL) and acetonitrile (7 mL) and added 200 mg active MS 4 Å and a solution of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (51.1 mg, 0.161 mmol) in 1 ml dry CH₃CN. The resulting mixture was stirred at rt for 30 min, followed by addition of 1H-tetrazole (43.4 mg, 0.620 mmol). The reaction stayed at rt for 1h and added tert-butyl hydroperoxide (5.0M in Decane) (0.074 mL, 0.372 mmol). The stirring continued for 1h and concentrated in vacuo. The residue was purified by column chromatography on silica gel, eluting with 0-8% MeOH/DCM to give the desired product. LCMS (ES, m/z): 922 [M+H]⁻

Step 2: 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-10-hydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide (90 mg, 0.098 mmol) and ammonia (7.0M in Me0H) (2 ml, 14.00 mmol) were sealed in a microwave tube. The mixture was heated to 50° C. and stirred for 4h. The reaction mixture was concentrated, and purified using mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) using a gradient solvent system with MeCN and 100 mM aqueous triethylammonium acetate to give three desired diastereomers. Lyophilization of the product fractions furnished 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one. LCMS (ES, m/z): 693 [M−H]⁻. ¹H NMR (H2O-d2, 500 MHz): δ_(H) 8.23 (2H, d, J=5.2 Hz), 7.81 (1H, s), 6.42 (1H, d, J=14.2 Hz), 5.99 (1H, d, J=8.6 Hz), 5.70 (1H, m), 5.40 (2H, m), 5.20 (1H, m), 4.62 (2H, m), 4.54 (1H, s), 4.15-4.30 (3H, m). ³¹P NMR: (H₂O-d₂, 202 MHz): δ −1.6, 52.1.

Example 290: 2-amino-9-[1(2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-15-hydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To (2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (Example 86, 5.0 mg, 0.0071 mmol) were added sodium phosphate buffer (pH 6.8, 50 mM, 0.5 mL) and adenosine monphosphate deaminase (3 mg). The reaction mixture was left to stir overnight, filtered, and purified by reverse phase HPLC (1-10% MeCN in aq NH₄HCO₃ (100 mM)) to afford the title compound as a diammonium salt. LCMS (ES, m/z): 690 [M−H]⁻. ¹H NMR (500 MHz, D₂O): δ 8.30 (s, 1H), 8.20 (s, 1H), 7.95 (s, 1H), 6.19 (s, 1H), 5.82 (d, J=6.0 Hz, 1H), 5.64 (m, 1H), 5.15 (m, 1H), 4.86 (d, J=4.5 Hz, 1H), 4.60 (m, 1H), 4.48-4.53 (m, 2H), 4.38 (m, 1H), 4.11 (dd, J=11.5, 4.0 Hz, 1H), 4.05 (m, 1H), 2.63 (m, 1H), 2.52 (m, 1H). ³¹P NMR: (202 MHz, D₂O): δ 55.78 (s), 52.44 (s).

Examples 291 through 348 as shown in Table 11 below, were prepared according to procedures analogous to those outlined in Example 290 above, from the starting compound (“St. Cmpd.”) indicated.

TABLE 11 Mass St. Ex. Structure Name [M − H]⁻ Cmpd. 291

9-[(5R,7R,8R,12aR,14R,15aS,16R)-7-(6- amino-9H-purin-9-yl)-2,10,16-trihydroxy- 2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-/][1,3,6,9,11,2,10] pentaoxadiphosphacyclotctradecin-14-yl]- 1,9-dihydro-6H-purin-6-one 642 3 292

9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-7 (6-amino-9H-purin-9-yl)-15-fluoro- 2,10,16-trihydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 1,9-dihydro-6H-purin-6-one 660 4 293

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fIuoro-2,10,16-trihydroxy-2,10-dioxido- 14-(6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 676 2 294

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR)-15- fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 660 21 295

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fluoro-2,10,16-trihydroxy-2,10-dioxido- 14-(6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 676 7 296

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 90 297

2-amino-9- [(2R,5R,7R,8R,10R,12aR,14R,15S,15aR, 16R)-15-fluoro-2,10,16-trihydroxy-14-(6- oxo-1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l)[1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 89 298

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 3) 708 91 299

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofurol]3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 4) 708 92 300

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16- fluoro-2,10,15-trihydroxy-14-(6-hydroxy- 9H-purin-9-yl)-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 676 5 301

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)- 2,10,15-trihydroxy-14-(6-hydroxy-9H- purin-9-yl)-16-methoxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 688 13 302

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16- azido-2,10,15-trihydroxy-14-(6-hydroxy- 9H-purin-9-yl)-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 699 14 303

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-10-oxido- 14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2- sulfidooctahydro-12H-5,8- methanofuro[3,2-/][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 694 143 304

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15- fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 708 81 305

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16- fluoro-2,10-dihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 692 120 306

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16- fluoro-2,10-dihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfidooclahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 2) 692 118 307

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15aS,16R)-16-fluoro-2,10- dihydroxy-14-(6-oxo-1,6-dihydro-9H- purin-9-yl)-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 3) 692 117 308

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16R)-15,16-difluoro- 2,10-dihydroxy-14-(6-oxo-1,6-dihydro- 9H-purin-9-yl)-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 247 309

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 2) 710 245 310

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 3) 710 246 311

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 4) 710 244 312

2-amino-9- [(5S,7R,8S,12aR,14R,15S,15aR,16R)-15- fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 660 25 313

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 2,10-dihydroxy-2,10-dioxido-14-(6-oxo- l,6-dihydro-9H-purin-9-yl)octahydro- 12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 642 27 314

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-2,10- dioxido-14-(6-oxo-1,6-dihydro-9H-purin- 9-yl)octahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 678 23 315

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dihydroxy-2,10- dioxido-14-(6-oxo-1,6-dihydro-9H-purin- 9-yl)octahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 678 26 316

2-amino-9- [(2aR,6aS,7R,8R,9aR,14R,14aS,15R)- 5,7,12-trihydroxy-5,12-dioxido-8-(6-oxo- 1,6-dihydro-9H-purin-9-yl)hexahydro- 6aH-2a,14-(epoxymethano)furo [3,2-d]oxeto[2,3-k][1,3,7,9,2,8] tetraoxadiphosphacyclotridecin- 15(2H,3H)-yl-1,9-dihydro-6H-purin-6- one 686 17 317

2-amino-9- [(2R,5S,7R,8R,10R,12aR,14R,15S,15aR)- 15-fluoro-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 692 130 318

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15- fluoro-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 2) 692 131 319

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16S)- 2,10,15,16-tetrahydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one 690 11 320

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 15-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,2,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 693 133 321

8-[(5R,7R,8R,12aR,14S,15S,15aS,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14- yl]pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one 674 54 322

2-amino-9-[(5R,7R,8R,,12aR,14R, 15S,15aR,16R)-15-chloro-2,10,16- trihydroxy-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)octahydro-12H- 5,8-methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 692 15 323

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16S)-15-fluoro-16- hydroxy-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro(3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 724 97 324

2-amino-9-[(5R,7R,8R,12aR,14R,15S, 15aR,16S)-15-fluoro-16-hydroxy-2,10- dioxido-14-(6-oxo-1,6-dihydro-9H-purin- 9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 724 99 325

2-amino-9-[(5R,7R,8R,12aR,14R,15S, 15aR,16S)-15-fIuoro-16-hydroxy-2,10- dioxido-14-(6-oxo-1,6-dihydro-9H-purin- 9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 724 96 326

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 711 130 327

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 711 128 328

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5.8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 3) 711 127 329

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15S,15aR,16S)-15,16-difluoro- 2,10-dioxido-14-(6-oxo-1,6-dihydro-9H- purin-9-yl)-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 144 330

2-amino-9-[(5R,7R,8S,12aR,14R,15S, 15aR,16R)-15,16-difluoro-2,10-dioxido- 14-(6-oxo-1,6-dihydro-9H-purin-9-yl)- 2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one (Diastereomer 2) 710 145 331

3-[(5R,7R,8S,12aR,14R,15S,15aR,16S)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 711 147 332

2-amino-9- [(5R,7R,8S,12aR,14R,15aS,16R)-16- fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9- yl)octahydro-l2H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one 660 22 333

9,9'-[(5R,7R,8S,12aR,14R,15R,15aS)- 2,10,15-trihydroxy-2,10- dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine-7,14- diyl]bis(1,9-dihydro-6H-purin-6-one) 643 50 334

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)- 10,15,16-trihydroxy-2,10-dioxido-14-(6- oxo-1,6-dihydro-9H-purin-9-yl)-2- sulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 690 38 335

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 710 260 336

7-[(5R,7R,8S,12aR,14R,15aS,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 16-fluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14- yl]imidazo[2,1-f][1,2,4]triazin-4(3H)-one (Diastereomer 1) 692 249 337

3-[(5R,7R,8S,12aR,14R,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 729 165 338

3-[(5R,7R,8S,12aR,14R,15aR,16R)-7-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 729 167 339

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-15- fluoro-2,16-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)-10- sulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one 708 266 340

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR)-15- fluoro-2,10-dioxido-14-(7-oxo-6,7- dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 694 181 341

5-amino-3- [(5S,7R,8R,12aR,14R,15S,15aR)-15- fluoro-2,10-dioxido-14-(7-oxo-6,7- dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 694 182 342

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 711 199 343

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 711 197 344

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dioxido-14-(7-oxo- 6,7-dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 712 200 345

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-2,10-dioxido-14-(7-oxo- 6,7-dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 712 201 346

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2-hydroxy-2,10- dioxido-10-sulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1) 695 279 347

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)- 7-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-15,16-difluoro-2-hydroxy-2,10- dioxido-10-sulfanyloctahydro-12H-5,8- methanofuro(3,2-l)[1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 695 280 348

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)- 15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10- disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 711 179

Biological Evaluation

The individual compounds described in the Examples are defined as STING agonists by demonstrating binding to the STING protein with an EC₅₀ of 20 uM or less in the STING Biochemical [³H]cGAMP Competition Assay (using either HAQ or wild type (WT) STING) and demonstrating interferon production with a 5% or greater luminescence induction at 30 uM in the INF-β secretion in the THP1 cell assay. The methods below describe each of these assays.

[³H]-cGAMP Synthesis

2.3 mL of buffer solution containing 80 mM tris Cl, 200 mM MgCl₂ and 20 mM NaCl followed by 0.32 mL of a 10 mM aq solution of GTP was added to a plastic 50 mL AMICON tube. A solution of [³H]ATP (21Ci/mmol, 45 mCi) in 0.5 mL H₂O was then added followed by 1 mL of a 1 mg/mL solution of DNA (Herring testes activator DNA, Sigma, #D6898) and 53 uL of a 47 mM solution of cGAS enzyme. Additional H₂O was added to bring the total volume to 10 mL.

The reaction was stirred for 2h at 37° C. and then added directly to an Amicon Ultra-15 10K centrifuge tube and spun for 1h at 4,000g. The collected solution was then purified on a semi-prep Mono Q column using the following mobile phases:

-   -   A: 0.05M TrisCl pH 8.5 adjusted with 1M NaOH     -   B: 0.05M TrisCl, 0.5M NaCl pH 8.5 adjusted with 1M NaOH

Gradient: 100% A for 5 min followed by a linear gradient to 50:50 (A:B) over 25 min, 3 mL/min, 254 nm.

The collected product fractions were pooled and adjusted to a total volume of 30 mL with buffer A. A total yield of 15.5 mCi of [³H]cGAMP was isolated at a radiochemical purity of 98.0% at a specific activity of 21.5Ci/mmol.

cGAS Enzyme

A recombinant DNA vector was chemically synthesized to express the truncated human cGAS enzyme (residues 161-522). To aid in expression and purification, the amino terminus contains a hexahistidine tag, SUMO tag and TEV cleavage site. The recombinant enzyme was overexpressed in ROSETTA™ 2(DE3) Single Competent Cells (Novagen). Affinity purification was carried out using HIS-Select HF Nickel Affinity Gel (Sigma) followed by size exclusion chromatography using a Hi-Load 26/60 SUPERDEX200 prep grade column (GE Healthcare). Fractions were pooled, concentrated, flash frozen in liquid nitrogen and stored at −80° C. until needed for research applications.

Example 349: ³H-cGAMP Filtration Binding Assay (HAQ STING)

The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni cell membranes (T. ni; Expression Systems, cat #94-002F, wwwv.expressionsystems.com) overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.

The basic HAQ STING filtration assay protocol is as follows:

The compounds were serially titrated by the Hamilton STARPlus CORE in a 96-well plate (Greiner, #651201) using a 1:3 ten-point dose response format. After compound preparation, a 2.2 ug/ml working concentration of STING membrane (SEQ. ID. No. 2) was prepared by diluting concentrated membrane into assay buffer (lx PBS; Invitrogen #SH30028.02) and douncing 7× using a manual tissue homogenizer (Wheaton, #357546). 148 uL of prepared membrane was then manually added to each well of a 96-well deep-well polypropylene plate (Fisher Scientific, #12-566-121). Following membrane addition, 2 uL of either titrated test compound, DMSO control (Sigma #276855), or cold cGAMP control (prepared in-house) was added to the appropriate wells using a Biomek FX. Compound and membrane then preincubated for 60 min at RT to allow compound binding to equilibrate. Following equilibration, 8 nM of [³H]c-GAMP ligand was prepared by diluting into assay buffer, and 50 uL of this working stock was then manually added to each well of the assay plate. Plates were then incubated at RT for 60 min, and the contents of each assay plate were then filtered through a 96-well GF/B filter plate (PerkinElmer, #6005250) using a TomTec MachIII Cell Harvester equipped with 20 mM HEPES buffer (Fisher Scientific, # BP299500). The filter plates were then dried at 55° C. for 30 min using a pressurized VWR oven before 30 uL of Ultima GoldF scintillate was added to each well. Tritium levels for each reaction well were then measured using a PerkinElmer TopCount plate reader.

After normalization to controls, the percent activity for each compound concentration was calculated by measuring the amount of remaining radioactivity. The plot of percent activity versus the log of compound concentration was fit with a 4-parameter dose response equation to calculate EC₅₀ values.

The final reaction conditions were:

Component Volume (uL) Final Concentration STING membrane 148 1.5 ug/ml ³H-cGAMP 50 2.0 nM Low Control (cold cGAMP) 2 10 uM Test compound/DMSO 2 10 uM

Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247, 0.082, 0.027, 0.009, 0.003, and 0.001 μM with 1.0% residual DMSO.

Full-Length STING (HAQ) Virus Generation

STING virus was generated using an insect cell baculovirus system. Spodoptera frugiperda Sf21 cells (Kempbio, Inc.) were diluted to 5e5 cells/ml in Sf-900II SFM media (LifeTechnologies #10902088) without antibiotics. The cell suspension was added to each well of a treated 6-well plate (2 mL per well, 1e6 cells total), and the cells were allowed to adhere for at least 30 min. Meanwhile, a 1 mL co-transfection mix was assembled by combining 500 ng of HAQ STING [STING(1-379)R71H,G230 Å,H232R,R293Q-GG-AviTag-GS-HRV3C-HIS8/pBAC1] DNA (Genewiz custom synthesis) with 1 mL Sf-900II SFM media containing 10 μL Cellfectin® II Reagent (Invitrogen #10362100) and 100 ng viral backbone BestBac 2.0, v-cath/chiA Deleted Linearized Baculovirus DNA (Expression Systems #91-002). The transfection mixtures were allowed to incubate for 30 min. After incubation, media was gently removed from the adhered cells in the 6-well plate, the 1 mL transfection mixtures were added (1 mL per well), and the plate was placed in a humidified incubator at 27° C. The following day, 1 mL Sf-900II SFM media (no antibiotics) was added to each well of the 6-well plate. After media addition, the cells were allowed to incubate with DNA (SEQ. ID. No. 3) at 27° C. for 5-7 days to generate the P0 viral stock. To generate P1 viral stocks, 0.5 mL of P0 viral supernatant was added to 50 mL uninfected Sf21 cells (seeded the day prior to infection at a density of 5×10⁵ cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5 g/mL gentamicin (Invitrogen #15710072). The infected cells were then incubated at 27° C. for 3d while shaking at 110 rpm (ATR Biotech Multitron Infors HT #AJ118). On day 3, P1 cultures were counted using a ViCell XR (Beckman Coulter Life Sciences #383556) to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 85-95%). Cultures were harvested in 50 mL conical tubes and centrifuged at 2000×g for 10 min at 4° C. The P1 viral supernatants were poured off into clean 50 ml centrifuge tubes, and the remaining P1 cell pellets were used to generate Baculovirus Infected Insect Cells (BIICs) according to in-house validated SOP. Cryopreservation media containing Sf-900II SFM media with 10% heat inactivated FBS, 10% DMSO (Sigma #D2650), and 5 μg/ml gentamicin was prepared in-house and sterilized through 0.22 μM filter immediately prior to use. P1 cell pellets were resuspended to a density of 2e7 cells/ml and aliquoted into cryovials (1 mL per vial). Cryovials were placed in Mr. Frosty cell freezers O/N at −80° C. and transferred to liquid nitrogen for long term storage the following day. To generate P2 viral stock, 0.5 mL of the P1 viral supernatant was added to 50 mL uninfected Sf21 cells (seeded the day prior to infection at a density of 5×10⁵ cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5 μg/mL gentamicin. These cells were incubated at 27° C. for 3d while shaking at 110 rpm before harvesting P2 stock with centrifugation at 2000×g for 10 min at 4° C. The P2 viral supernatants were poured off and discarded, while the P2 cell pellets were used to generate P2 BIICs following the same protocol described above. The baculovirus generation protocol has been validated to consistently produce P1/P2 BIICs with titers of 2e9 pfu/mL (2e7 cells/mL×100 pfu/cell).

Full-Length STING (HAQ) Expression

To generate STING membranes, P1/P2 BIICs were amplified overnight by adding thawed BIICs to Sf21 cells seeded at a density of 1.0×10⁶ cells/mL. The volume of BIIC used to infect the culture was calculated using an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in the overnight amplification. After culturing overnight, the cells were counted on a ViCell XR to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 80-90%). The volume of infected Sf21 cells from the overnight amplification used to infect the large-scale expression of Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, www.expressionsystems.com) seeded at a density of 1.0×10⁶ in cell media (ESF921 SFM containing 5 μg/mL gentamicin) at MOI=2.0 was calculated based on (100 pfu/infected Sf21 cell). The cells were allowed to express for 48h at 27° C. before harvesting the cell pellet, by centrifugation at 3,400×g for 10 min at 4° C. T. ni cells were counted on a ViCell XR to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 80-90%) prior to harvest.

Full-Length STING (HAQ) Membrane Generation

Buffer stock reagents:

-   -   1) 1 M HEPES pH 7.5, Teknova, Cat#H1035     -   2) 5 M NaCl, Sigma Aldrich, Cat#S5150-1 L     -   3) KCl, Sigma Aldrich, Cat#319309-500ML     -   4) Complete EDTA-free protease inhibitor tablets, Roche         Diagnostics, Cat#11873580001     -   5) Benzonase, Universal Nuclease, Pierce, Cat#88702

Lysis buffer [25 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl, (Benzonase 1:5000, Complete Protease Inhibitor tab/50 mL)] was added to the pellet of cells expressing full-length STING (HAQ) prepared above at 5 mL Lysis buffer/g of cell pellet. The pellet was resuspended and dounced twenty times using a Wheaton Dounce Homogenizer to disrupt the cell membrane. Homogenized lysate was then passed through the emulsiflex-C₅ microfluidizer at a pressure close to 5000 PSI. The resuspended pellet was centrifuged at 36,000 rpm (100,000×g) in a 45Ti rotor in the ultra-high speed centrifuge for 45 min, 4° C. The supernatant was removed. The pellet then was resuspended in wash buffer [(25 mM HEPES pH7.5, 1 mM MgCl₂, 20 mM KCl, 1M NaCl (Complete Protease Inhibitor tab/50 mL)] at a volume of 50 mL pellet/centrifuge tube. The pellet/wash buffer mixture was then homogenized, using a glass homogenizer on ice (20 strokes), followed by centrifugation at 36,000 rpm for 45 min at 4° C. The supernatant was removed. The wash step was repeated once more. The resulting membrane was resuspended in 20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, EDTA-free Protease Inhibitors (1 tablet/50 mL). The protein concentration was measured by Bradford assay (Bio-Rad Protein Assay, Cat#500-0006), and protein enrichment was determined by SDS-PAGE and confirmed by Western blot. The resuspended membranes were stored at −80° C.

Full-Length HAQ STING [STING(1-379)R71H, G230 Å,H232R, R293Q-GG-AviTag-GS-HRV3C-HIS8]Amino Acid Sequence.

(SEQ. ID. No. 2) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLH LASLQLGLLLNGVCSLAEELHHIHSRYRGSYWRTVRACLGCPLRRGALLL LSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEK GNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYI LLPLDCGVPDNLSMADPNIRFLDKLPQQTADRAGIKDRVYSNSIYELLEN GQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCQTLEDILA DAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA VPSTSTMSQEPELLISGMEKPLPLRTDFSGGGLNDIFEAQKIEWHEGSLE VLFQGPHHHHHHHH Full-length HAQ [STING(1-379)R71H, G230 Å,H232R,R293Q-GG-AviTag-GS-HRV3C-HIS8/pBAC1] Plasmid DNA Sequence:

(SEQ. ID. No. 3) GGAACGGCTCCGCCCACTATTAATGAAATTAAAAATTCCAATTTTAAAAA ACGCAGCAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAA ATGTCGTCGACATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAA AAAATATTGAACGATTTGAAAGAAAACAATGTACCGCGCGGCGGTATGTA CAGGAAGAGGTTTATACTAAACTGTTACATTGCAAACGTGGTTTCGTGTG CCAAGTGTGAAAACCGATGTTTAATCAAGGCTCTGACGCATTTCTACAAC CACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTTAATCAAATCCCA AGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGC TCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTAT TGAATAATAAAACAATTATAAATGCTAAATTTGTTTTTTATTAACGATAC AAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGC GTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCAC AGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTG TCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAA ATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAA ATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTA CCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAG TTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATT TGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCT TCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTC CAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTA TACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTA ATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATA GTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCG CAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAA CCTATAAATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCC GTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTG CCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTC CGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAA CGGGGTCTGCAGCCTGGCTGAGGAGCTGCACCACATCCACTCCAGGTACC GGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTGCCCCCTCCGC CGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCAAA TGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGC AGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATC TCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATG GTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCC GGATTCGAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTG AGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGTGCCTGATAA CCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCCCAGC AGACCGCTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATC TATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTA CGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTG GCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACA CTTGAGGACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCT CATTGCCTACCAGGAACCTGCAGATGACAGCAGCTTCTCGCTGTCCCAGG AGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGTGGGC AGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCC TGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTCCGCACGGATT TCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGAATGG CATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCA CCATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACT AACCTAGGTAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTT ATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACG TATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGC GAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAAT CCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGT TTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCAC AAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTT GTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCT TTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAA CACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTAT TAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCC GAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAA CACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATT GCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCA GACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGG CAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCG GTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGC GGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGT CGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCG GTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGT TGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGG AGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTG GAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATT TGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGC CGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTG GTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATA AGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTC AATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGATCGATCC CGCACGCCGATAACAAGCCTTTTCATTTTTACTACAGCATTGTAGTGGCG AGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCTCACTGACTCG CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTT ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCA TAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTG CTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCC TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCC ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG CGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGA AGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGC CACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAG CTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATC GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTA ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTC TATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAA AAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAA CGTTTACAATTTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG CGATCGGTGCGGGCCTCTTCGCTATTACGCCA

Certain compounds of the disclosure were evaluated in HAQ STING in vitro binding assay as described above. The following table tabulates the biological data for these compounds as EC₅₀ values.

TABLE 12 ³H-cGAMP filtration binding assay for HAQ STING Compound EC₅₀ (nM) Compound EC₅₀ (nM) Compound EC₅₀ (nM) Example 1 <1 Example 55 55.3 Example 134 <1 Example 2 1.3 Example 56 89.4 Example 135 253.4 Example 3 257.1 Example 57 437.9 Example 136 3.7 Example 4 22 Example 58 3.1 Example 137 11.4 Example 5 1.1 Example 59 11.8 Example 138 738.8 Example 6 <1 Example 60 42.5 Example 139 <1 Example 7 1.2 Example 61 18.2 Example 140 12.9 Example 8 13.6 Example 66 117.1 Example 141 <1 Example 9 7.2 Example 67 6.4 Example 142 6.2 Example 10 4.9 Example 68 63.8 Example 143 1.05 Example 11 1.2 Example 69 <1 Example 144 <1 Example 12 <1 Example 70 2 Example 145 <1 Example 13 1.5 Example 71 <1 Example 146 5.6 Example 14 <1 Example 72 <1 Example 147 1.4 Example 15 378.1 Example 73 834 Example 148 33.3 Example 16 12.4 Example 74 5.8 Example 149 8.4 Example 17 121 Example 75 2.6 Example 150 <1 Example 18 53.9 Example 76 75.4 Example 151 <1 Example 19 173 Example 77 160.5 Example 152 <1 Example 20 <1 Example 78 14.6 Example 244 156.6 Example 21 1.3 Example 79 103.2 Example 245 2.7 Example 22 4822 Example 80 1.3 Example 246 21.3 Example 23 <1 Example 81 <1 Example 247 <1 Example 24 2.4 Example 82 21.5 Example 290 2.5 Example 25 3.4 Example 83 3.7 Example 291 64.7 Example 26 <1 Example 85 <1 Example 292 2.6 Example 27 1.3 Example 86 <1 Example 293 1.9 Example 28 18.7 Example 87 <1 Example 294 6.6 Example 30 91 Example 88 <1 Example 295 29.6 Example 31 94 Example 89 8272 Example 296 4.1 Example 32 11.1 Example 90 1.7 Example 297 <1 Example 33 11.9 Example 91 15.8 Example 298 52.9 Example 34 24.6 Example 92 114 Example 299 1325 Example 35 7.9 Example 93 <1 Example 300 5.1 Example 36 21.5 Example 94 24.6 Example 301 4.8 Example 37 619.6 Example 95 13.9 Example 302 2.3 Example 38 3 Example 96 5.3 Example 303 <1 Example 69 <1 Example 97 <1 Example 304 3.3 Example 70 2 Example 98 1.8 Example 305 326.2 Example 71 <1 Example 99 <1 Example 306 3.6 Example 72 <1 Example 100 16.7 Example 307 <1 Example 73 834 Example 101 8 Example 308 1.6 Example 74 5.8 Example 102 <1 Example 309 4 Example 39 24.4 Example 117 1 Example 310 27.6 Example 40 3.2 Example 118 1.4 Example 311 1595 Example 41 11.8 Example 119 17.8 Example 312 122.5 Example 42 15.2 Example 120 45.1 Example 313 210.6 Example 43 162.2 Example 121 <1 Example 314 <1 Example 44 17.1 Example 123 5.1 Example 315 49.9 Example 45 24.8 Example 124 <1 Example 316 2324 Example 46 11.7 Example 125 21.7 Example 317 <1 Example 47 39.2 Example 126 9.7 Example 318 6.2 Example 48 1.2 Example 127 24.4 Example 319 7.3 Example 49 30 Example 128 <1 Example 320 6.3 Example 50 2.7 Example 129 88.6 Example 321 112.2 Example 51 287.1 Example 130 <1 Example 322 68.8 Example 52 1.5 Example 131 2.4 Example 332 8.2 Example 53 1.7 Example 132 25.9 Example 333 45.9 Example 54 <1 Example 133 61.4

Example 350: ³H-cGAMP Filtration Binding Assay (WT STING)

The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni cell membranes (T. ni; Expression Systems, cat #94-002F, www.expressionsystems.com) overexpressing full-length WT STING prepared in-house and tritiated cGAMP ligand also purified in-house.

The basic WT STING filtration assay protocol is as follows:

16 nM of [³H]c-GAMP ligand was prepared by diluting into assay buffer, and 50 uL of this working stock was manually added to each well of the assay plate. After ligand addition, 2 uL of either titrated test compound, DMSO control (Sigma #276855), or cold cGAMP control (prepared in-house) was added to the appropriate wells using a Biomek FX. The serially titrated compound was prepared on a Hamilton STARPlus CORE in a 96-well plate (Greiner, #651201) using a 1:3 ten-point dose response format. Following compound addition, a 2.2 ug/ml working concentration of STING membrane (SEQ. ID. No. 4) was prepared by diluting concentrated membrane into assay buffer (lx PBS; Invitrogen # SH30028.02) and douncing 7× using a manual tissue homogenizer (Wheaton, #357546). 148 uL of this prepared membrane was then manually added to each well of a 96-well deep-well polypropylene plate (Fisher Scientific, #12-566-121). Compound, ligand, and membrane then incubated for 60 min at RT before the contents of each assay plate were filtered through a 96-well GF/B filter plate (PerkinElmer, #6005250) using a TomTec MachIII Cell Harvester equipped with 20 mM HEPES buffer (Fisher Scientific, # BP299500). The filter plates were then dried at 55° C. for 30 min using a pressurized VWR oven before 30 uL of Ultima GoldF scintillate was added to each well. Tritium levels for each reaction well were then measured using a PerkinElmer TopCount plate reader.

After normalization to controls, the percent activity for each compound concentration was calculated by measuring the amount of remaining radioactivity. The plot of percent activity versus the log of compound concentration was fit with a 4-parameter dose response equation to calculate EC₅₀ values.

The final reaction conditions were:

Component Volume (uL) Final Concentration STING membrane 148 1.5 ug/ml ³H-cGAMP 50 4.0 nM Low Control (cold cGAMP) 2 10 uM Test compound/DMSO 2 10 uM

Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247, 0.082, 0.027, 0.009, 0.003, and 0.001 μM with 1.0% residual DMSO.

Full-Length STING (WT) Virus Generation

STING virus was generated using an insect cell baculovirus system. Spodoptera frugiperda Sf21 cells (Kempbio, Inc.) were diluted to 5e5 cells/ml in Sf-900II SFM media (LifeTechnologies #10902088) without antibiotics. The cell suspension was added to each well of a treated 6-well plate (2 mL per well, 1e6 cells total), and the cells were allowed to adhere for at least 30 min. Meanwhile, a 1 mL co-transfection mix was assembled by combining 500 ng of WT STING[STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBAC1] (Genewiz custom synthesis) with 1 mL Sf-900II SFM media containing 10 μL Cellfectin® II Reagent (Invitrogen #10362100) and 100 ng viral backbone BestBac 2.0, v-cath/chiA Deleted Linearized Baculovirus DNA (Expression Systems #91-002). The transfection mixtures were allowed to incubate for 30 min. After incubation, media was gently removed from the adhered cells in the 6-well plate, the 1 mL transfection mixtures were added (1 mL per well), and the plate was placed in a humidified incubator at 27° C. The following day, 1 mL Sf-900II SFM media (no antibiotics) was added to each well of the 6-well plate. After media addition, the cells were allowed to incubate with DNA [(SEQ. ID. No. 5) and linearized viral backbone BestBac 2.0] at 27° C. for 5-7 days to generate the P0 viral stock. To generate P1 viral stocks, 0.5 mL of P0 viral supernatant was added to 50 mL uninfected Sf21 cells (seeded the day prior to infection at a density of 5×10⁵ cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5 g/mL gentamicin (Invitrogen #15710072). The infected cells were then incubated at 27° C. for 3d while shaking at 110 rpm (ATR Biotech Multitron Infors HT #AJ118). On day 3, P1 cultures were counted using a ViCell XR (Beckman Coulter Life Sciences #383556) to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 85-95%). Cultures were harvested in 50 mL conical tubes and centrifuged at 2000×g for 10 min at 4° C. The P1 viral supernatants were poured off into clean 50 ml centrifuge tubes, and the remaining P1 cell pellets were used to generate Baculovirus Infected Insect Cells (BIICs) according to in-house validated SOP. Cryopreservation media containing Sf-900II SFM media with 10% heat inactivated FBS, 10% DMSO (Sigma #D2650), and 5 μg/ml gentamicin was prepared in-house and sterilized through 0.22 μM filter immediately prior to use. P1 cell pellets were resuspended to a density of 2e7 cells/ml and aliquoted into cryovials (1 mL per vial). Cryovials were placed in Mr. Frosty cell freezers O/N at −80° C. and transferred to liquid nitrogen for long term storage the following day. To generate P2 viral stock, 0.5 mL of the P1 viral supernatant was added to 50 mL uninfected Sf21 cells (seeded the day prior to infection at a density of 5×10⁵ cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5 μg/mL gentamicin. These cells were incubated at 27° C. for 3d while shaking at 110 rpm before harvesting P2 stock with centrifugation at 2000×g for 10 min at 4° C. The P2 viral supernatants were poured off and discarded, while the P2 cell pellets were used to generate P2 BIICs following the same protocol described above. The baculovirus generation protocol has been validated to consistently produce P1/P2 BIICs with titers of 2e9 pfu/mL (2e7 cells/mL×100 pfu/cell).

Full-Length STING (WT) Expression

To generate STING membranes, P1/P2 BIICs were amplified overnight by adding thawed BIICs to Sf21 cells seeded at a density of 1.0×10⁶ cells/mL. The volume of BIIC used to infect the culture was calculated using an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in the overnight amplification. After culturing overnight, the cells were counted on a ViCell XR to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 80-90%). The volume of infected Sf21 cells from the overnight amplification used to infect the large-scale expression of Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, www.expressionsystems.com) seeded at a density of 1.0×10⁶ in cell media (ESF921 SFM containing 5 μg/mL gentamicin) at MOI=2.0 was calculated based on (100 pfu/infected Sf21 cell). The cells were allowed to express for 48h at 27° C. before harvesting the cell pellet, by centrifugation at 3,400×g for 10 min at 4° C. T. ni cells were counted on a ViCell XR to confirm infection had occurred (cell size ≥3 μm larger than uninfected cells and viability approximately 80-90%) prior to harvest.

Full-Length STING (WT) Membrane Generation

Buffer stock reagents:

-   -   1) 1 M HEPES pH 7.5, Teknova, Cat#H1035     -   2) 5 M NaCl, Sigma Aldrich, Cat#S5150-1 L     -   3) KCl, Sigma Aldrich, Cat#319309-500ML     -   4) Complete EDTA-free protease inhibitor tablets, Roche         Diagnostics, Cat#11873580001     -   5) Benzonase, Universal Nuclease, Pierce, Cat#88702

Lysis buffer [25 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl, (Benzonase 1:5000, Complete Protease Inhibitor tab/50 mL)] was added to the pellet of cells expressing full-length STING (WT) prepared above at 5 mL Lysis buffer/g of cell pellet. The pellet was resuspended and dounced twenty times using a Wheaton Dounce Homogenizer to disrupt the cell membrane. Homogenized lysate was then passed through the emulsiflex-C5 microfluidizer at a pressure close to 5000 PSI. The resuspended pellet was centrifuged at 36,000 rpm (100,000×g) in a 45Ti rotor in the ultra-high speed centrifuge for 45 min, 4° C. The supernatant was removed. The pellet then was resuspended in wash buffer [(25 mM HEPES pH 7.5, 1 mM MgCl₂, 20 mM KCl, 1M NaCl (Complete Protease Inhibitor tab/50 mL)] at a volume of 50 mL/pellet/centrifuge tube. The pellet/Wash buffer mixture was then homogenized, using a glass homogenizer on ice (20 strokes), followed by centrifugation at 36,000 rpm for 45 min at 4° C. The supernatant was removed. The wash step was repeated once more. The resulting membrane was resuspended in 20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, EDTA-free Protease Inhibitors (1 tablet/50 mL). The protein concentration was measured by Bradford assay (Bio-Rad Protein Assay, Cat#500-0006), and protein enrichment was determined by SDS-PAGE and confirmed by Western blot. The resuspended membranes were stored at −80° C.

Full-Length STING WT [STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8] Amino Acid Sequence:

(SEQ. ID. No. 4) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLH LASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLL LSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEK GNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYI LLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLEN GQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILA DAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA VPSTSTMSQEPELLISGMEKPLPLRTDFSGGGLNDIFEAQKIEWHEGSLE VLFQGHHHHHHHH

Full-Length WT STING [STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBAC1] Plasmid Sequence:

(SEQ. ID. No. 5) GGAACGGCTCCGCCCACTATTAATGAAATTAAAAATTCCAATTTTAAAAA ACGCAGCAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAA ATGTCGTCGACATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAA AAAATATTGAACGATTTGAAAGAAAACAATGTACCGCGCGGCGGTATGTA CAGGAAGAGGTTTATACTAAACTGTTACATTGCAAACGTGGTTTCGTGTG CCAAGTGTGAAAACCGATGTTTAATCAAGGCTCTGACGCATTTCTACAAC CACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTTAATCAAATCCCA AGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGC TCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTAT TGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATAC AAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGC GTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCAC AGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTG TCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAA ATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAA ATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTA CCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAG TTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATT TGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCT TCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTC CAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTA TACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTA ATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATA GTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCG CAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAA CCTATAAATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCC GTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTG CCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTC CGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAA CGGGGTCTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACC GGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTGCCCCCTCCGC CGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCAAA TGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGC AGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATC TCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATG GTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCC GGATTCGAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTG AGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGTGCCTGATAA CCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCCCAGC AGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATC TATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTA CGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTG GCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCGGACA CTTGAGGACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCT CATTGCCTACCAGGAACCTGCAGATGACAGCAGCTTCTCGCTGTCCCAGG AGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGTGGGC AGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCC TGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTCCGCACGGATT TCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGAATGG CATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCA CCATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACT AACCTAGGTAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTT ATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACG TATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGC GAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAAT CCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGT TTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCAC AAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTT GTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCT TTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAA CACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTAT TAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCC GAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAA CACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATT GCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCA GACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGG CAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCG GTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGC GGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGT CGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCG GTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGT TGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGG AGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTG GAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATT TGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGC CGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTG GTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATA AGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTC AATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGATCGATCC CGCACGCCGATAACAAGCCTTTTCATTTTTACTACAGCATTGTAGTGGCG AGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCTCACTGACTCG CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTT ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCA TAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTG CTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCC TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCC ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG CGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGA AGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGC CACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAG CTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATC GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTA ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTC TATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAA AAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAA CGTTTACAATTTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG CGATCGGTGCGGGCCTCTTCGCTATTACGCCA

Certain compounds of the disclosure were evaluated in WT STING in vitro binding assay as described above. The following table tabulates the biological data for these compounds as EC₅₀ values.

TABLE 13 ³H-cGAMP filtration binding assay for WT STING Compound EC₅₀ (nM) Compound EC₅₀ (nM) Compound EC₅₀ (nM) Example 1 3.5 Example 139 1.2 Example 226 27.8 Example 2 0.7 Example 141 <1 Example 227 260.6 Example 3 574.3 Example 142 174.8 Example 228 1005 Example 4 43.5 Example 144 4.6 Example 229 31% Inh @ 20 uM Example 5 1.5 Example 145 14 Example 230 382.4 Example 6 2.9 Example 148 1932 Example 231 540.4 Example 7 <1 Example 149 110.8 Example 232 3283 Example 8 81.4 Example 152 5.7 Example 233 33.8 Example 9 12.4 Example 153 6.5 Example 234 275.2 Example 10 23.2 Example 154 60 Example 235 14.9 Example 13 0.7 Example 155 <1 Example 236 6963 Example 14 1.4 Example 156 <1 Example 237 307.2 Example 20 1.4 Example 157 <1 Example 238 66.3 Example 21 1.4 Example 158 40 Example 239 3.2 Example 25 16.1 Example 159 7.1 Example 240 73.6 Example 26 3.7 Example 160 10.3 Example 241 471.8 Example 27 10 Example 161 6.2 Example 242 1.1 Example 28 264.9 Example 162 100.7 Example 243 26.3 Example 29 5835 Example 163 14.3 Example 247 1.1 Example 32 25 Example 164 312.4 Example 248 31.7 Example 38 24.9 Example 165 2.9 Example 249 <1 Example 41 505.9 Example 166 463.2 Example 250 17.3 Example 42 219 Example 167 87.7 Example 251 <1 Example 43 1015 Example 168 8.6 Example 252 768.4 Example 44 1363 Example 169 186.6 Example 253 54.5 Example 45 104.8 Example 170 25.5 Example 254 39.7 Example 46 337 Example 171 18.7 Example 255 301.9 Example 47 444.1 Example 172 105.9 Example 256 93.5 Example 51 995.8 Example 176 16.2 Example 257 135.9 Example 61 378.7 Example 177 7.9 Example 258 5 Example 62 2.6 Example 178 37.4 Example 259 1.5 Example 63 68.8 Example 179 111.7 Example 260 7.2 Example 64 61.6 Example 180 832.1 Example 261 21.8 Example 65 249.6 Example 181 459.2 Example 262 57.3 Example 70 19.4 Example 182 3276 Example 263 4.8 Example 71 4.4 Example 183 3180 Example 264 177.5 Example 72 <1 Example 184 <1 Example 265 5473 Example 81 3.1 Example 185 201.1 Example 266 1.9 Example 82 14.6 Example 186 30.2 Example 267 6267 Example 84 6.3 Example 187 6.5 Example 268 77 Example 86 2 Example 188 47.6 Example 269 4837 Example 89 <1 Example 189 423.9 Example 270 4383 Example 90 4.4 Example 190 408.7 Example 271 12.8 Example 93 1.9 Example 191 281.2 Example 272 749.6 Example 94 523.8 Example 192 3867 Example 273 216.4 Example 95 64 Example 193 190.5 Example 274 2.3 Example 96 57.8 Example 194 159.2 Example 275 21.8 Example 97 0.4 Example 195 1855 Example 276 321.5 Example 98 151.7 Example 196 1033 Example 277 28.7 Example 99 1.8 Example 197 1.2 Example 278 477.9 Example 100 552 Example 198 19.1 Example 279 1235 Example 101 74.4 Example 199 72 Example 280 1186 Example 84 6.3 Example 200 15 Example 281 2485 Example 86 2 Example 201 649.5 Example 282 12190 Example 102 1 Example 202 813.5 Example 283 17.2 Example 103 <1 Example 203 7.3 Example 284 128.3 Example 104 11.6 Example 204 1233 Example 285 277.1 Example 105 3.9 Example 205 42.7 Example 286 4010 Example 106 <1 Example 206 259.7 Example 287 80.2 Example 107 17.9 Example 207 1.4 Example 288 24.9 Example 108 1784 Example 208 23.3 Example 289 5.3 Example 109 1393 Example 209 9.5 Example 308 3.3 Example 110 141.4 Example 210 28.5 Example 323 3.7 Example 111 155.1 Example 211 1045 Example 324 10.7 Example 112 1279 Example 212 11.8 Example 325 866.1 Example 113 44.2 Example 213 135 Example 326 89.7 Example 114 3.3 Example 214 2.1 Example 327 14 Example 115 67.9 Example 215 33.1 Example 328 74.4 Example 116 1624 Example 216 3.4 Example 329 31.7 Example 117 1.7 Example 217 66.2 Example 330 204 Example 118 1.6 Example 218 13.8 Example 331 182.9 Example 119 40 Example 219 530.5 Example 334 110.5 Example 120 393.7 Example 220 29% Inh Example 335 5.2 @ 20 uM Example 122 179.2 Example 221 <1 Example 336 422.2 Example 124 4 Example 222 60 Example 337 29.2 Example 126 447.4 Example 223 302.5 Example 338 441.7 Example 128 19.5 Example 224 37.4 Example 129 973.3 Example 225 779.3

Example 351: IFN-β Secretion in THP1 Cell Culture (5h)

The ability of compounds to stimulate the secretion of interferon-beta from THP1 cells was measured using a human IFN-β AlphaLISA kit (Perkin Elmer, Cat. No. AL265F). The basic protocol is as follows:

A Labcyte Echo 550 acoustic dispenser was used to transfer 120 nL of compound dissolved in DMSO into the wells of an empty, sterile 384-well microplate, (Corning, Cat. No. 3712). THP1 cells (American Type Culture Collection, Cat. No. TIB202) previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and immediately diluted 10-fold into 37° C. assay medium (RPMI 1640+L-Glutamine & phenol red, Life Technologies, Cat. No. 11875-085; 0.5% heat inactivated fetal bovine serum, Sigma Aldrich, Cat. No. F4135; 1 mM Sodium Pyruvate, Life Technologies, Cat. No. 11360-070; lx non-essential amino acids; Life Technologies, Cat. No. 11140-050). The cell viability and count was ascertained using a Beckman Coulter V-Cell XR cell counter. The cells suspension was centrifuged at 200×g for 5 min at RT. Cells were resuspended to a density of 0.8×10⁶/mL in 37° C. assay medium. Subsequent liquid transfers were performed using either a Matrix electronic multichannel pipette or an Agilent Bravo Automated Liquid Handling Platform.

The assay was started by dispensing 40 μL of the previously prepared cell suspension into the wells of the plate containing compounds. After 5h incubation at 37° C., 5% CO₂ in a humidified atmosphere, the plate of cells and compounds was centrifuged at 200×g for 5 min at RT. From each well, 5 μL of supernatant was transferred into corresponding wells of a white 384-well plate (Perkin Elmer, Cat. No. 6005620). To these supernatant-containing wells was added 10 μL of 5× Anti-Analyte Acceptor beads (50 μg/mL of AlphaLISA HiBlock Buffer) and incubated for 30 min at RT while shaking on an orbital plate shaker. To each well was added 10 μL of 5× Biotinylated Antibody Anti-analyte (5 nM in AlphaLISA HiBlock Buffer) and incubated on an orbital plate shaker for 60 min at RT or overnight at 4° C. To each well was added 25 μL of 2×SA-Donor beads (80 μg/mL in AlphaLISA HiBlock Buffer) and incubated for 30-45 min at RT in the dark while shaking on an orbital plate shaker. The plate was then read on a Perkin Elmer Envision (λ_(ex)=680 nm, λ_(em)=570 nm). The percent effect of the AlphaLISA signal at each compound concentration was calculated based on 30 uM cGAMP positive controls and 0.3% DMSO negative controls. The plot of percent effect versus the log of compound concentration was fit with a 4-parameter concentration response equation to calculate EC₅₀ values. The test compounds were tested at concentrations 30000, 10000, 3333, 1111, 370.4, 123.4, 41.2, 13.7, 4.6, and 1.5 nM with 0.3% residual DMSO. The control compound, cGAMP was tested at concentrations 100000, 33333, 11111, 3704, 1235, 412, 137, 46, and 15 nM with 0.3% residual DMSO.

Compounds of the disclosure were evaluated for IFN-β secretion in THP1 cell culture as described above. The following table tabulates the biological data for these compounds as percent activation relative to 2′3′-cGAMP at the 30 μM concentration.

TABLE 14 IFN-β secretion in THP1 cell culture (5 h) % Effect at 30 μM Compound relative to 2′3′-cGAMP Example 1 154 Example 2 148 Example 3 149 Example 4 171 Example 5 152 Example 6 114 Example 7 112 Example 8 98 Example 9 143 Example 10 126 Example 11 169 Example 12 89 Example 13 39 Example 14 139 Example 15 39 Example 16 57 Example 17 81 Example 18 74 Example 19 49 Example 20 159 Example 21 142 Example 22 109 Example 23 125 Example 24 90 Example 25 145 Example 26 150 Example 27 164 Example 28 60 Example 29 25 Example 30 131 Example 31 100 Example 32 125 Example 33 200 Example 34 125 Example 35 51 Example 36 120 Example 37 57 Example 38 123 Example 39 74 Example 40 77 Example 41 8 Example 42 101 Example 43 130 Example 44 135 Example 45 161 Example 46 143 Example 47 141 Example 48 104 Example 49 74 Example 50 85 Example 51 36 Example 52 143 Example 53 127 Example 54 83 Example 55 83 Example 56 64 Example 57 7 Example 58 92 Example 59 76 Example 60 12 Example 61 34 Example 62 68 Example 63 83 Example 64 93 Example 65 87 Example 66 54 Example 67 111 Example 68 72 Example 69 31 Example 70 100 Example 71 113 Example 72 102 Example 73 5 Example 74 57 Example 75 96 Example 76 39 Example 77 39 Example 78 162 Example 79 145 Example 80 234 Example 81 138 Example 82 111 Example 83 133 Example 84 128 Example 85 154 Example 86 137 Example 87 99 Example 88 54 Example 89 119 Example 90 117 Example 91 106 Example 92 62 Example 93 155 Example 94 138 Example 95 118 Example 96 67 Example 97 113 Example 98 54 Example 99 135 Example 100 35 Example 101 129 Example 102 132 Example 103 114 Example 104 154 Example 105 137 Example 106 208 Example 107 171 Example 108 15 Example 109 151 Example 110 181 Example 111 200 Example 112 33 Example 113 154 Example 114 163 Example 115 177 Example 116 149 Example 117 211 Example 118 174 Example 119 151 Example 120 72 Example 121 108 Example 122 75 Example 123 128 Example 124 128 Example 125 138 Example 126 167 Example 127 145 Example 128 146 Example 129 33 Example 130 215 Example 131 238 Example 132 150 Example 133 117 Example 134 138 Example 135 43 Example 136 131 Example 137 124 Example 138 51 Example 139 110 Example 140 96 Example 141 111 Example 142 111 Example 143 157 Example 144 114 Example 145 141 Example 146 59 Example 147 146 Example 148 13 Example 149 8 Example 150 89 Example 151 57 Example 152 16 Example 153 234 Example 154 198 Example 155 139 Example 156 119 Example 157 151 Example 158 123 Example 159 140 Example 160 12 Example 161 130 Example 162 116 Example 163 127 Example 164 41 Example 165 245 Example 166 143 Example 167 278 Example 168 164 Example 169 117 Example 170 134 Example 171 142 Example 172 26 Example 173 142 Example 174 5 Example 175 120 Example 176 114 Example 177 180 Example 178 111 Example 179 66 Example 180 143 Example 181 178 Example 182 47 Example 183 23 Example 184 108 Example 185 61 Example 186 179 Example 187 134 Example 188 251 Example 189 93 Example 190 200 Example 191 516 Example 192 50 Example 193 399 Example 194 258 Example 195 61 Example 196 14 Example 197 237 Example 198 205 Example 199 139 Example 200 219 Example 201 222 Example 202 171 Example 203 194 Example 204 101 Example 205 132 Example 206 166 Example 207 189 Example 208 163 Example 209 170 Example 210 178 Example 211 114 Example 212 285 Example 213 255 Example 214 224 Example 215 277 Example 216 170 Example 217 70 Example 218 152 Example 219 142 Example 220 6 Example 221 405 Example 222 171 Example 223 11 Example 224 288 Example 225 467 Example 226 439 Example 227 290 Example 228 6 Example 229 6 Example 230 116 Example 231 189 Example 232 21 Example 233 254 Example 234 75 Example 235 262 Example 236 5 Example 237 222 Example 238 183 Example 239 164 Example 240 141 Example 241 300 Example 242 156 Example 243 165 Example 244 96 Example 245 234 Example 246 217 Example 247 166 Example 248 42 Example 249 106 Example 250 6 Example 251 175 Example 252 23 Example 253 73 Example 254 131 Example 255 49 Example 256 149 Example 257 8 Example 258 120 Example 259 91 Example 260 113 Example 261 90 Example 262 108 Example 263 114 Example 264 123 Example 265 25 Example 266 168 Example 267 5 Example 268 181 Example 269 109 Example 270 235 Example 271 229 Example 272 44 Example 273 85 Example 274 124 Example 275 157 Example 276 45 Example 277 174 Example 278 68 Example 279 160 Example 280 191 Example 281 8 Example 282 39 Example 283 288 Example 284 260 Example 285 195 Example 286 16 Example 287 202 Example 288 153 Example 289 58 Example 290 90 Example 291 98 Example 292 114 Example 293 86 Example 294 84 Example 295 70 Example 296 92 Example 297 176 Example 298 121 Example 299 5 Example 300 86 Example 301 107 Example 302 103 Example 303 106 Example 304 104 Example 305 50 Example 306 153 Example 307 174 Example 308 132 Example 309 232 Example 310 117 Example 311 9 Example 312 90 Example 313 117 Example 314 143 Example 315 92 Example 316 12 Example 317 107 Example 318 150 Example 319 143 Example 320 115 Example 321 36 Example 322 41 Example 323 92 Example 324 99 Example 325 10 Example 326 34 Example 327 126 Example 328 99 Example 329 105 Example 330 67 Example 331 116 Example 332 113 Example 333 10 Example 334 66 Example 335 60 Example 336 85 Example 337 204 Example 338 219 Example 339 175 Example 340 306 Example 341 7 Example 342 23 Example 343 255 Example 344 337 Example 345 144 Example 346 182 Example 347 225 Example 348 8

It will be appreciated that various of the above-discussed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. 

1-10. (canceled)
 11. A compound of formula (I′):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of —O— and —S—; X^(a) and X^(a1) are each independently selected from the group consisting of O, and S; X^(b) and X^(b1) are each independently selected from the group consisting of O, and S; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R⁴ and R⁵ are connected to form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionally R⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and with the provisos that (a) when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

then R⁵ and R³ are not both selected from the group consisting of H, F and OH; and (b) the compound is not selected from the group consisting of

and pharmaceutically acceptable salt thereof.
 12. The compound selected from a compound according to claim 11 and pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected from the group consisting of —O— and —S—; X^(a) and X^(a1) are each independently selected from the group consisting of O and S; X^(b) and X^(b1) are each independently selected from the group consisting of O and S; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R¹ and R^(1a) are each H; R² and R^(2a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected from the group consisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; R⁶ and R^(6a) are each independently selected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆ haloalkyl, where said R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a) are each H; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; and optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position.
 13. The compound selected from a compound according to claim 11 and pharmaceutically acceptable salts thereof, wherein the compound of formula (I′) is a compound of formula (I′b):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R^(1a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R^(1a)C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, OH, CN, and N₃; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵ are not both selected from the group consisting of OH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.
 14. The compound selected from a compound according to claim 11 pharmaceutically acceptable salts thereof, wherein the compound of formula (I′) is a compound of formula (I′c):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3 substituents R¹⁰, where each R¹⁰ is independently selected from the group consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆ cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, and N(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, Cl, Br, I, and OH; each R⁹ is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position.
 15. A compound of formula (I″):

or a pharmaceutically acceptable salts thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; and optionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position; and with the provisos that (a) when Y and Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² are each selected from the group consisting of

then R⁵ and R³ are not both selected from the group consisting of H, F and OH, and (b) the compound is not selected from the group consisting of

and pharmaceutically acceptable salts thereof.
 16. The compound according to claim 15, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I″) is a compound of formula (I″a):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; and each R⁹ is independently selected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, —O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl.
 17. The compound according to claim 16, or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R^(2a) is F; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; and each R⁹ is independently H.
 18. The compound according to claim 15, wherein R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.
 19. The compound according to claim 15, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I″) is a compound of formula (I″b):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1) are each independently selected from the group consisting of OR⁹ and SR⁹; X^(d) and X^(d1) are each independently selected from the group consisting of O and S; R³ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently H; and R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.
 20. The compound according to claim 19, or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base² are each independently selected from the group consisting of


21. The compound according to claim 15, wherein at least one of Base¹ and Base² are each independently selected from the group consisting of


22. A compound is selected from the group consisting of:

and pharmaceutically acceptable salt thereof.
 23. The compound according to claim 22, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 24. The compound according to claim 22, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 25. A pharmaceutical composition, said pharmaceutical composition comprising: (a) a compound selected from the group consisting of a compound according to claim 11 and pharmaceutically acceptable salts thereof and (b) a pharmaceutically acceptable carrier.
 26. A method of inducing an immune response in a subject, said method comprising administering a therapeutically effective amount of a compound selected from the group consisting of a compound according to claim 11 and pharmaceutically acceptable salts thereof to the subject.
 27. A method of inducing an immune response in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 25 to the subject.
 28. A method of inducing a STING-dependent type I interferon production in a subject, said method comprising administering a therapeutically effective amount of a compound selected from the group consisting of a compound according to claim 11 and pharmaceutically acceptable salts thereof to the subject.
 29. A method of inducing a STING-dependent type I interferon production in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 25 to the subject. 30-33. (canceled) 